Bispecific molecule binding TLR9 and CD32 and comprising a T cell epitope for treatment of allergies

ABSTRACT

A molecule or molecule complex capable of binding to TLR9 and to CD32 comprising at least one epitope of at least one antigen, and its use a medicament for the treatment of allergies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §120 andis a continuation in part of U.S. patent application Ser. No.12/281,504, filed on Sep. 3, 2008 and entitled BISPECIFIC MOLECULEBINDING TLR9 AND CD32 AND COMPRISING A T CELL EPITOPE FOR TREATMENT OFALLERGIES, which is the U.S. national stage of International ApplicationNo. PCT/EP2007/001722, filed Feb. 28, 2007, which claims the benefit ofpriority under 35 U.S.C. §119 from European Patent Application No.06110672.0, filed Mar. 3, 2006 and entitled NOVEL MOLECULES. Thecontents of the foregoing patent applications are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The entire content of a Sequence Listing titled “Sequence_Listing.txt,”created on May 28, 2013 and having a size of 69 kilobytes, which hasbeen submitted in electronic form in connection with the presentapplication, is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to molecules with binding specificity toboth, Toll-like Receptor 9 (TLR9) and CD32 containing one or more T cellantigen epitopes. The invention further relates to the production ofthese molecules and their use for the preparation of medicaments for thetreatment of allergies.

BACKGROUND

Allergy is considered to be a hypersensitive reaction to proteins in theenvironment (air/water/food). Allergens are antigens to which atopicpatients respond with IgE antibody responses subsequently leading toallergic reactions. Antigens in the complexes or fusion proteins can beenvironmental allergens (e.g. house dust mite, birch pollen, grasspollen, cat antigens, cockroach antigens), or food allergens (e.g. cowmilk, peanut, shrimp, soya), or a combination of both. IgE molecules areimportant because of their role in effector cell (mast cell, basophilesand eosinophils) activation. More recently, it has been accepted thatIgE also plays an important role in the induction phase of allergicdiseases, by up-regulating the antigen capture potential of B cells anddendritic cells (DC), both through low affinity (CD23) and high affinityreceptors (FcεRI) [1]. The negative functions of IgE antibodies can becounteracted by allergen specific IgG antibodies. e.g. because theydirect the immune response away from B cells to monocytes and DC [2]. Inaddition, they compete with IgE molecules for allergen binding sites.Allergies therefore can be treated, cured and prevented by the inductionof allergen specific IgG molecules.

IgG molecules have a serum half-life of approximately 3 weeks ascompared to roughly 3 days for IgE molecules. IgE molecules are inducedby the interaction between (naive) B cells and Th2 cells which providethe IL-4 and IL-13 together with CD40L expression necessary to induce aclass switch to IgE in memory B cells and plasma cells [3]. In contrast,TM cells, which produce IFN-γ and IL-2, induce a class switch to IgG.Therefore, induction of Th1, rather than Th2 helper T cell responsesagainst allergens, is beneficial for the prevention, treatment and cureof allergic diseases.

To date several forms of active vaccination using allergens are used.The most common is the so called “Immunotherapy”, which depends onfrequent immunizations with relatively high concentrations of allergens.This technique is only moderately effective in a minority of allergicdiseases such as Bee venom allergy and in some cases of Rhinitis andConjunctivitis, and recently some reports have shown effectiveness inasthma and atopic dermatitis. More recently rush immunotherapy, whereincreasing amounts of allergen are injected in a rather short timeframe, has been proposed with slightly better results [4; 5]. Usuallythe subcutaneous route is used for administration of the allergens, butrecently this route has been compared to oral application or even localapplication, the results are generally positive but not alwaysconsistent. A different technique for immunotherapy is the one describedby Saint-Remy (EP 0 178 085 and 0 287 361), which makes use ofautologous IgG antibodies which are in vitro complexed to the relevantallergens. This technique allows far smaller amounts of allergen to beapplied with fewer side effects.

The mechanism behind these therapies is unclear. In the classicaltherapy there seems to be a beneficial effect if the therapy induces anincrease in specific IgG antibodies, although not every significantincrease of specific IgG is correlated with successful immunotherapy. Apossible argument why this is the case is the relatively low affinity ofIgG antibodies for CD32 on B cells, monocytes and mast cells. TheSaint-Remy approach selects the specific IgG antibodies from thepatient, which are subsequently mixed with relevant allergens in vitro.This way they assure that the allergen cannot react freely with cells orother antibody isotypes on cells such as IgE on mast cells. In additionthey claim that anti-idiotypic antibodies are raised against thespecific IgG molecules, which in the future will prevent allergy.

In WO 97/07218 Allergen-anti-CD32 Fusion Proteins are described. In thispublication the problems with isolating specific IgG molecules and thelow affinity of these IgG antibodies for CD32 are circumvented and therisk factors of classical immunotherapy, which uses complete “IgEbinding” allergens, are reduced. However, the claimed induction of Th1memory responses due to solely directing the anti-CD32 containingvaccine to dendritic cells cannot be substantiated.

Even in view of the intensive research for therapeutic approaches totreat allergic diseases, there is still a great demand for providingmedicaments for successful treatment of allergies.

The object of the invention is therefore to provide novel molecules withimproved properties for the treatment of allergic diseases.

According to the invention this object is achieved by the subject matterof the claims.

BRIEF SUMMARY OF THE INVENTION

CD32 is strongly expressed on monocytes/dendritic cells and B cells andthus the molecule of the present invention is designed to direct theimmune response to these important immunological cells, with theintention to prevent allergen presentation by the B cells, whilepromoting allergen presentation by especially dendritic cells (DCs), thelatter leads to induction of Th1 responses against the allergens in themolecule or molecule complex that can be formulated as vaccine. Morerecent knowledge shows that two types of dendritic cells (DC) exist:myeloid (mDC) and plasmacytoid dendritic cells (pDC) [6], which has ledto the new concept of DC1 and DC2 cells [7]. In this concept DC1 cellspromote the induction of Th1 cell development after antigen specificstimulation and DC2 cells support the development of Th2 cells. Monocytederived DC (or mDC) are generally considered to be of DC1 type, whereaspDC are considered to be DC2 type [6]. Both types of DC express CD32aand will induce an allergen specific T cell response; however it is notguaranteed that the outcome will be of Th1 type. In fact, in allergicdonors Th2 responses are more likely [8]. Importantly, the pDC expressthe TLR9 receptor, which binds CpG-ODNs (oligodeoxynucleotides [ODNs]containing unmethylated CpG motifs). Activation of this receptor in thepDC leads to a very strong production of IFN-α and IL-12 [9], whichpromotes Th1 induction and thus transforms the potential DC2 into DC1cells.

Therefore, the molecule of the invention can combine the activation ofthe TLR9 receptor in pDC with the specific stimulation and induction ofallergen specific Th1 cells and comprises therefore a significantimprovement of earlier concepts.

The invention comprises a molecule or a molecule complex having bindingspecificity for toll-like receptor 9 and CD32, wherein the molecule ormolecule complex includes at least one epitope, preferably at least oneT cell epitope, of at least one antigen. The molecule or moleculecomplex of the invention will also bypass the effector function of mastcells, which carry IgE, for the native allergen of which T cell epitopeshave been selected to be part of the fusion protein.

Preferably the molecule or molecule complex according to the inventioncan have one or more of the following unique characteristics:

-   -   Activation and induction of allergen specific Th1 cells, without        activation of allergen-specific B-cells.    -   Activation and induction of allergen specific Th1 cells, without        activation of mast cells or any other effector cell, which, by        means of allergen specific IgE or IgG, may become activated by        the natural allergens of which the selected T cell epitopes are        represented in the molecule or molecule complex of the        invention.

The CD32-binding part of the molecule or molecule complex of theinvention selects the relevant cells, which should internalize thecomplete molecule or molecule complex.

After internalization of the fusion protein according to the presentinvention by antigen presenting cells the molecule of the invention isdegraded and various peptides, in eluding the incorporated T cellepitope(s) are presented on the MHC class Il molecules of the antigenpresenting cells, therefore stimulating allergen specific T cells.

The incorporated TLR9-binding structure(s) in the molecule or moleculecomplex of the invention are necessary for the induction of an allergenspecific Th1 memory pool, by binding to the cytoplasmatic [10; 11] TLR9receptor. Activation of the TLR9 receptor leads to a strong induction ofIFN-α and IL12 production [9].

According to the present invention, a molecule is a single entity madeup of atoms and/or other molecules by covalent bonds. The molecule canbe made up of one single class of substances or a combination thereof.Classes of substances are e.g. polypeptides, carbohydrates, lipids,nucleic acids etc.

A molecule complex is an aggregate of molecules specifically andstrongly interacting with each other. A complex of various molecules maybe formed by hydrophobic interactions (such as e.g. the binding ofantibody variable regions in an Fv) or by strong binding of one moleculeto another via ligand/receptor interactions such as antibody-antigenbinding or avidin-biotin or by complex formation via chelating chemicalgroups and the like. The molecule complex is preferably produced throughchemical conjugation, recombinant fusion and/or affinity binding.

An antigen can be a structure which can be recognized by an antibody, aB-cell-receptor or a T-cell-receptor when presented by MHC class I or Ilmolecules.

An epitope is the smallest structure to be specifically bound within byan antibody, a B-cell-receptor or a T-cell receptor when presented byMHC class I or Il molecules. Specificity is defined as preferred bindingto a certain molecular structure (in antibody/antigen interactions alsocalled epitope) within a certain context.

A domain is a discrete region found in a protein or polypeptide. Amonomer domain forms a native three-dimensional structure in solution inthe absence of flanking native amino acid sequences. Domains of theinvention will specifically bind to CD32 and/or TLR9 and/or display orpresent epitopes. Domains may be used as single domains or monomerdomains or combined to form dimers and multimeric domains. For example,a polypeptide that forms a three-dimensional structure that binds to atarget molecule is a monomer domain.

According to the present invention the term antibody includes antibodiesor antibody derivatives or fragments thereof as well as relatedmolecules of the immunoglobulin superfamily (such as soluble T-cellreceptors). Among the antibody fragments are functional equivalents orhomologues of antibodies including any polypeptide comprising animmunoglobulin binding domain or a small mutated immunoglobulin domainor peptides mimicking this binding domain together with an Fc region ora region homologous to an Fc region or at least part of it. Chimericmolecules comprising an immunoglobulin binding domain, or equivalents,fused to another polypeptide are included.

Allergens are antigens to which atopic patients respond with allergicreactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of an experiment in which anautoimmune response was induced in mice using ScFV-1-coil and mAb IV.3.

FIG. 2 is a graph showing IFN-α production by human pDCs afterstimulation with CPG-C.

FIG. 3 is another graph showing IFN-α production of human pDCs afterstimulation with CPG-C.

FIG. 4 is a graph showing IL-6 and TNFα production of pDCS afterstimulation with CPG-C.

FIG. 5 is a graph showing the production of IgG antibodies in responseto various immunogens.

FIG. 6 is a graph of T cell proliferation in response to variousimmunogens.

FIG. 7 is a graph of cytokine production in response to variousimmunogens.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a molecule or a molecule complex being capable ofbinding to toll-like receptor 9 (TLR9) and Fc gamma receptor RII (CD32)and including at least one epitope of at least one antigen.

In one embodiment of the invention the molecule or molecule complexcomprises at least three parts, one part being a structure specificallybinding to TLR9 (monovalently, bivalently or multivalently), anotherpart being a structure specifically binding to CD32 (monovalently,bivalently or multivalently) and at least one other part being one ormore T cells epitopes of an antigen and/or allergen. The parts may beindependent structures which are linked together either by chemicallinkages or by genetic fusion or by other (non-covalent) interactionssuch as ligand-receptor or antibody interactions.

The linkages between the different parts may be different. For example,in one preferred embodiment, the linkage between the parts binding toTLR9 and CD32 is by genetic fusion and the link to at least one of the Tcell epitopes is via a receptor/ligand interaction (e.g.biotin-streptavidin). The advantage of such a setup is the flexibilityin production. The bispecific (anti-TLR9/anti-CD32), generic part of themolecule complex can be produced in the same way for all patients,selected T cell epitopes are linked to the generic part of the moleculecomplex according to the need. The selection can be based on diseaseprevalence or on results of individual specificity tests of patients(specific allergy). The complex formation may be performed centralizedor at the bed side or at a physician's office.

Chemical linkage of molecules of the various binding molecules of thesame or different chemical class may be achieved by many differenttechniques yielding either a defined molecular ratio of the variousparts of the molecule or molecule complex of the invention. It may alsolead to a mixture of molecules with different molecular ratios of thevarious parts of the molecule or molecule complex of the invention.

The ratio of the various parts of the invention may be an equimolar ornon-equimolar. The molecule may be monovalent for binding to TLR9 and/orCD32 and/or T cell epitope(s). It may also be bi-, tri- and multivalentfor at least one of the parts of the molecule or the molecule complex.If the binding to TLR9 and/or CD32 is bivalent or of higher valency, thebinding specificity may be for one or for more epitopes on CD32 and/orTLR9 respectively.

In another embodiment of the invention the binding specificities of themolecule are overlapping so that one part of the molecule or themolecule complex of the invention is binding to both, TLR9 and CD32.Such a part could be selected for by simultaneous screening of moleculesfor binding to both CD32 and TLR9 or by engineering of a molecule tobind both, CD32 and TLR9. For example, a protein scaffold can be usedfor displaying loops to bind CD32 and other loops that bind to TLR9.

In a further embodiment of the invention, a protein scaffold can be usedto display structures that bind CD32, structures that bind TLR9 and todisplay T-cell epitopes.

The specific binding molecules can be natural ligands for CD32 and TLR9and derivatives thereof. For example, the Fc-part of immunoglobulin isbinding to CD32. CpG is a naturally occurring ligand for TLR9.

The specific binding molecules can be peptides. CD32- and TLR9-specificpeptides according to the invention can be selected by various methodssuch as phage display technology or by screening of combinatorialpeptide libraries or peptide arrays. The peptides can be selected andused in various formats such as linear, constrained or cyclic peptides,the peptides can be chemically modified for stability and/orspecificity.

A specifically binding peptide may also be derived from analysis ofinteraction of a naturally occurring proteinaceous ligand to TLR9 andCD32 by isolation of the minimal binding site of the ligand.

The specific binding peptides can be used as such in the molecule or themolecule complex of the invention or used to be incorporated into otherstructures such as by grafting into protein scaffolds, antibodies andprotein domains or chemically coupled to carrier molecules which mightbe part of the molecule or molecule complex of the invention.

The binding part of the molecules or molecule complex of the inventioncan be comprised of proteins such as antibodies or antibody fragments(such as Fab, Fv, scFv, dAb, F(ab)₂, minibody, small mutatedimmunoglobulin domains, soluble T-cell receptor, etc.). Antibodies andantibody fragments and derivatives may be generated and selected forbinding to TLR9 and/or CD32 according to known methods such as hybridomatechnology, B-cell cloning, phage display, ribosome display or cellsurface display of antibody libraries, array screening of variantantibodies.

The binding parts of the molecules or molecule complexes of theinvention can be protein domains which occur naturally or domains whichare artificially modified. Protein domains or domain derivatives, e.g.domains with mutations such as amino acid substitutions, deletions orinsertions or chemically modified domains may be selected for binding toTLR9 and/or CD32 according to known methods (e.g. phage and cell surfacedisplay of libraries of domains or domain variants and screening, arraysof variant molecules and screening). The domains include but are notlimited to molecules from the following classes:

-   -   EGF-like domain, a Kringle-domain, a fibronectin type I domain,        a fibronectin type Il domain, a fibronectin type III domain, a        PAN domain, a GIa domain, a SRCR domain, a Kunitz/Bovine        pancreatic trypsin Inhibitor domain, a Kazal-type serine        protease inhibitor domain, a Trefoil (P-type) domain, a von        Willebrand factor type C domain, an Anaphylatoxin-like domain, a        CUB domain, a thyroglobulin type I repeat, a LDL-receptor class        A domain, a Sushi domain, a Link domain, a Thrombospondin type I        domain, an immunoglobulin domain, an Immunoglobulin-like domain,        a C-type lectin domain, a MAM domain, a von Willebrand factor        type A domain, an A-domain, a Somatomedin B domain, a WAP-type        four disulfide core domain, an F5/8 type C domain, a Hemopexin        domain, an SH2 domain, an SH3 domain, a Laminin-type EGF-like        domain, a CTLA-4 domain, a C2 domain.

In a preferred embodiment, the binding part of a molecule or moleculecomplex of the invention comprises a small mutated immunoglobulin domain(SMID) as described in PCT/EP2006/050059.

The binding part of the molecule or molecule complex of the inventioncan be nucleic acids such as RNAs or DNAs which can be selected forspecific binding to TLR9 and/or CD32 according to known methods such asaptamer screening and in vitro evolution techniques.

It is contemplated that also other molecule classes will be able to showspecific binding to TLR9 and or CD32. Libraries of other chemicalentities than the ones mentioned above, including carbohydrates, lipids,and small organic molecules, may be screened for specific binding toTLR9 and/or CD32 and may be incorporated into the molecule or moleculecomplex of the invention.

A preferred embodiment of the invention is a recombinant fusion proteinconsisting of at least one epitope of at least one antigen, at least onebinding site interacting with TLR9 and at least one binding siteinteracting with CD32. The antigen can be as small as one T cell epitopefrom one antigen or can be a cocktail or mixture of one or more T cellepitopes from one or more different antigens fused or linked together ina way that allows proper processing and presentation by MHC molecules.The order of the epitopes can be selected according to differentcriteria such as product stability effective processing,(non-)recognition by preformed antibodies in the treated persons.Generally one will select for a stable molecule which can be efficientlypresented by MHC and which will lead to minimal recognition by preformedantibodies.

The invention further comprises the physical coupling of at least onemolecule interacting with TLR9, at least one molecule interacting withCD32 and one or more T cell epitopes from one or more antigens linkedtogether in a random form.

Additionally, the invention provides the preparation of a medicamentcontaining the fusion protein according to the invention and its use fortreatment of allergies. The medicament can be a vaccine formulationcontaining the molecule or molecule complex according to the invention,useful esp. for active immunotherapy.

The recombinant production of bispecific binding structures of themolecule or the molecule complex of the invention (i.e. binding to CD32and to TLR9) can be accomplished in different ways, e.g. by:

-   -   Quadroma technology (fused hybridomas) [12; 13];    -   bispecific scFvs, either as “diabodies” or simply by genetic        fusion of different scFvs [14];    -   single-domain antibodies in which VH recognizes one antigen and        VL another one;    -   chi-bAbs (as described in EP0640130);    -   small mutated immunoglobulin domains, by including engineered        immunoglobulin domains, specifically binding to CD32 and/or to        TLR9 in constructs coding either for complete antibodies or for        antibody fragments such as Fab (according to PCT/EP2006/050059);    -   in the context of this invention, binding to CD32 can also be        accomplished by monomeric or multimeric immunoglobulin Fc        region(s) or a parts thereof especially when the affinity for        CD32 of the Fc parts is enhanced, while TLR9 binding is achieved        through the normal binding site (VH/VL) of the antibody;    -   Fc-region(s) of an immunoglobulin or parts thereof, binding to        CD32, fused to any other TLR9 specific binding motif;    -   the Fc part of the above mentioned antibody may be        “glyco-engineered” to increase the affinity for human FcγR's        [15] engineered scaffolds, specifically binding to TLR9 and/or        CD32 of any kind can be used and linked together as needed.        These binding scaffolds can be protein domains, fibronectin III,        lipocalins, Protein A or α-amylase inhibitor, Ankyrin Repeat        Proteins, a C2 domain, an A-domain, an EGFR like domain, a dab,        a chi-bAb, CTLA-4, gamma crystalline or any other protein,        protein domain or part thereof.

The molecule or molecule complex of the invention consist of one or moreepitopes and one or more binding structures, which interact with TLR9,preferably human TLR9 and one or more binding structures, which interactwith CD32, preferably human CD32. For easy in vivo testing of theinventive protein the binding structures that recognize human TLR9 andhuman CD32 may cross react with monkey or mouse TLR9 and monkey or mouseCD32. The selected antigens/allergens may be complete natural/nativeproteins or parts of these, as long as epitopes which can be presentedon MHC class Il molecules and which can be recognized by T cells arepresent on the sequences present in the molecule or molecule complex.The part(s) of the molecule or molecule complex, which interact withTLR9 and CD32 may be complete or incomplete (modified) antibodies orfragments or derivatives thereof, as long as binding to TLR9 and CD32 isretained.

Alternatively, anti-TLR9 and anti-CD32 antibodies or derivatives orfragments thereof, which still specifically recognize and bind to humanTLR9 and CD32 such as expressed by B cells, and dendritic cells can beused.

Alternatively, the antibodies interacting with TLR9 or CD32 are improvedantibodies with higher affinity than the original antibodies.

Exemplary antibody molecules are intact immunoglobulin molecules andthose portions of an immunoglobulin molecule that contains the paratope,including those portions known as Fab, Fab′, F(ab′)₂, Fc and F(v), dAb.

The antibodies can be IgG, IgM, IgE, IgA or IgD. The moleculesinteracting with TLR9 or CD32 can be of any origin, preferably ofmammalian origin, preferably of human, mouse, camel, dog or cat originor humanized. Preferably the molecules are antibodies, preferably humanor humanized antibodies.

As used herein, if the molecule or molecule complex of the invention isa fusion protein, it can be expressed in host cells which cover any kindof cellular system which can be modified to express the fusion protein.Within the scope of the invention, the term “cells” means individualcells, tissues, organs, insect cells, avian cells, mammalian cells,hybridoma cells, primary cells, continuous cell lines, stem cells and/orgenetically engineered cells, such as recombinant cells expressing anantibody according to the invention.

Cells can be bacterial cells, fungal cells yeast cells, insect cells,fish cells and plant cells. Preferably the cells are animal cells, morepreferably mammalian cells. These can be for example BSC-1 cells, LLC-MKcells, CV-1 cells, CHO cells, COS cells, PerC6 cells, murine cells,human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells,MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells,WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0, NSO cells orderivatives thereof.

Preferably the binding structures of the molecule or the moleculecomplex of the invention recognizing TLR9 and CD32 are small mutatedimmunoglobulin domains, being for example an Fab fragment in which onebinding site (either specific for CD32 or for TLR9) is formed by VH/VL,and is combined with a second binding site (either specific for TLR9 orfor CD32 respectively) which can be an engineered CL or an engineeredCH1, CH2, CH3, CH4, VL or VH domain according to PCT/EP/2006/050059; ora complete antibody in which one binding site is formed by VH/VL, and iscombined with a second binding site which can be an engineered CL, CH1,CH2, CH3, CH4, VL or VH domain according to PCT/EP/2006/050059.

According to the invention, the molecule or molecule complex contains atleast one structure that specifically binds to CD32.

An anti-CD32 antibody can be derived by known methods (such as hybridomatechnology, B-cell cloning and antibody library screening). Forselection, cells displaying CD32 in a natural format can be used or arecombinant extracellular part of CD32 can be used or synthetic peptidesselected from the CD32 amino acid sequence can be used. Selectioncriteria are that the binding structure recognizes CD32a. In case alsoCD32b is recognized it is preferred that the affinity for CD32a≧CD32b.

As an example, the Fab fragment from the anti-CD32 IV.3 antibody derivedfrom the cell line HB-217 can be used. Using the method e.g. describedby Orlandi et al 16, the Fab fragment is cloned from the cell lineHB-217. Alternatively, other formats such as scFv can be constructed ofthe known V-gene sequences. However, for optimal combination with ananti-TLR9 antibody or Fab fragment or Fv fragment it is preferred toselect specific binders using one or more of the small mutatedimmunoglobulin domain libraries from CH1, CH2 CH3, CH4, CL, VL or VH.

Selected CH1, CH2, CH3, CH4, CL1 VL or VH domains can then be clonedinto the existing sequence of an anti-TLR9 antibody or a Fab or an Fvfragment thereof thus generating a bi-specific antibody or Fab fragment.

The selected CD32 binding entities should preferably have the followingcharacteristics:

1. Interaction with CD32a leads to internalization of thereceptor-binding-structure complex, activation of the antigen presentingcell through the ITAM motif in the cytoplasmic tail of the receptor andantigen presentation of the linked/fused T cell epitopes;

2. Interaction with CD32b leads to negative signaling of the receptorthrough the ITIM motif;

3. Interaction should show cross reactivity between human and monkeyCD32 (for testing of efficacy in a relevant in vivo model);

4. Interaction should show cross reactivity with mouse CD32 (for testingin an established in vivo model for allergy);

For obtaining a binding structure that specifically binds to TLR9,several procedures can be used (such as hybridoma technology, B-cellcloning and antibody library screening). For selection, cells expressingTLR9 in a natural format can be used to isolate natural TLR9 or arecombinant TLR9 can be used or synthetic peptides selected from theTLR9 amino acid sequence can be used. Alternatively, purified TLR9 orTLR9 expressed by cell lines can be used. Antibody genes coding for VLand VH respectively can be extracted after selection for binding to TLR9and be used to design a recombinant antibody or Fab fragment specificfor human TLR9. Alternatively, a single-chain Fv can also be made andfused with the anti-CD32 scFv mentioned above. However, for optimalcombination with the anti-CD32 antibody or scFv or Fab fragment it ispreferred to select specific binders using one or more of the smallmutated immunoglobulin domain libraries from CH1, CH2 CH3, CH4, CL, VHor VL. Selected CH1, CH2, CH3, CH4, CL, VL or VH domains can then becloned into the existing antibody or Fab fragment or scFv of anti-CD32antibody thus generating a bi-specific Fab fragment. The selected TLR9binding entities should preferably have the following characteristics:

1. Interaction with TLR9 leads to signal transduction and cytokineproduction;

2. Interaction may show cross reactivity between human and monkey TLR9(for testing of efficacy in a relevant in vivo model);

3. Interaction may show cross reactivity with mouse TLR9 (for testing inan established in vivo model for allergy) and CD32.

Of course the fusion protein can similarly be made using the Fab part ofan existing aTRL9 monoclonal antibody. Using the method e.g. describedby Orlandi et a116, the Fab fragment is cloned from e.g. clone 26C593available from Imgenex Corp., as described above for the fab fragment ofthe aCD32 Ab IV.3. Again for optimal combination with the anti-TLR9Fabfragment it is best to select specific binders for CD32 using one ormore of the small mutated immunoglobulin domain libraries from CH1, CH2,CH3, CH4, CL, VL or VH. Selected CH1, CH2, CH3, CH4, CL, VL or VHdomains can then be cloned into the existing Fab fragment of anti-TLR9antibody thus generating a bi-specific molecule.

Finally, e.g. in the absence of available suitable existing Ab's forboth CD32 and TLR9, it is also possible to construct a bi-specificmolecule using the small mutated immunoglobulin domain libraries fromCH1, CH2 CH3 or CL to select specific binders for both CD32 and TLR9which are subsequently combined to form new structures existing of atleast 1 binding structure specific for CD32 and 1 binding structurespecific for TLR9 derived from any of the possible libraries in any ofthe possible combinations (CH1-CH1 or CH1-CH2 or CH1 CH3 or CH2-CH4, orCH3-CH4, or CH1-CH4 or CH2-CH3 etc.).

Alternatively, a single variable domain of the immunoglobulinsuperfamily may be selected for binding to TLR9 or CD32 with CDR-loops.The selected binder is then randomized at non-structural loop positionsto generate a library of variable domains which is selected for therespective other antigen, i.e. in case of a variable domain binding withCDR loops to TLR9 the selection is for binding to CD32 and vice versa.It is also possible to select a library of a V-domain which containsvariations in the CDR loops at the same time as variations in thenon-CDR-loops for binding to TLR9 and CD32 sequentially orsimultaneously.

Such bispecific V-domains may also be part of antibodies or antibodyfragments such as single-chain-Fvs, Fabs or complete antibodies.

Selection of a suitable TLR9 epitope, i.e. sequence 244-256 (SEQ IDNo 1) of the mature TLR9 protein in amino acid 1 letter code:

CPRHFP QLHPDTFS 214    250    257

will fulfill criterion 1 and 2 but not 3, whereas sequence 176-191 (SEQID No 2) of the mature protein TLR9 in amino acid 1 letter code:

LTHL SLKYNNLTVV PR 176  180        191

and

Sequence 216-240 of the mature protein TLR9 (SEQ ID No 3) in amino acid1 letter code:

ANLT ALRVLDVGGN CRRCDHAPNP C 216  220        230        240will fulfill all three criteria and are thus preferred for use in thisinvention.

The process for producing the molecule or molecule complex is carriedout according to known methods, e.g. by using recombinant cloningtechniques or by chemical cross linking.

A product as described in this invention can be produced in thefollowing way:

The obtained VH and VL of the anti-CD32 antibody are fused to CH 1 andCL respectively. The CL has previously been engineered using SMIDtechnology (PCT/EP2006/050059) and selected using phage display to bindto TLR9 as described below. CH1 is fused at its C-terminus to a sequenceencoding the selected T cell epitopes. These two fusion-protein encodinggenes are cloned into an expression vector allowing the expression oftwo independent genes (or into two independent expression vectors) andare co-expressed in bacteria, yeast or animal cells or any othersuitable expression system. Thus, an Fab with the desiredcharacteristics, i.e. binding to CD32, binding to TLR9 and carrying therelevant T-cell epitopes is produced.

Alternative examples applying SMID technology include:

-   -   An scFv against TLR9 is derived from a phage display library or        from an existing hybridoma, and a CD32 binding molecule is        derived from a CH2-CH4, or CH3-CH4, or CH1-CH4 or small mutated        immunoglobulin domain library. These two coding sequences are        ligated together and a sequence coding for T cell epitopes is        attached. The fusion protein is then expressed in bacteria,        yeast or animal cells or any other suitable expression system;    -   Alternatively, TLR9-specificity and CD32-specificity are        swapped: An scFv against CD32 is derived e.g. from a phage        display library or from an existing hybridoma, and a        TLR9-binding molecule is derived from a CH2-CH4, or CH3-CH4, or        CH1-CH4 or small mutated immunoglobulin domain library. These        two coding sequences are ligated together and a sequence coding        for T cell epitopes is attached. The fusion protein is then        expressed in bacteria, yeast or animal cells or any other        suitable expression system;    -   VH and VL of an anti-TLR9 antibody are fused to CH 1 and CL        respectively. CL has previously been engineered and selected        using phage display to bind to CD32 (SMID). CH1 is fused at its        C-terminus to a sequence encoding the T cell epitopes. These two        fusion-protein encoding genes are cloned into an expression        vector allowing the expression of two independent genes (or into        two independent expression vectors) and are coexpressed in        bacteria, yeast or animal cells or any other suitable expression        system, (again, anti-TLR9 and anti-CD32 can be swapped. CH1 and        CL can also be swapped);    -   Heavy and light chain genes of an anti-TLR9 antibody are taken        as a whole. In the heavy chain gene, the CH2 (or CH1 or CH3 or        CH4) region is replaced by a CH2 (or CH1 or CH3 or CH4 or CL or        VH or VL) region which has previously been engineered and        selected using phage display to bind to CD32 (small mutated        immunoglobulin domain). CH1, CH2, CH3 or CH4 is fused at its        C-terminus to a sequence encoding the T cell epitopes. These two        genes are again cloned in expression vectors and expressed in        animal cells;    -   2 small mutated immunoglobulin domains, one specific for TLR9,        the other specific for CD32 are fused and combined with T-cell        epitopes;    -   1 small mutated immunoglobulin domain with 2 different        specificities (TLR9 and CD32) is combined with T-cell epitopes.

Antigens and Epitopes:

The antigens that are part of the molecule or molecule complex accordingto the invention can be complete allergens, denatured allergens or anyantigens that are treated in any possible way to prevent binding to IgE.Such treatment may consist of epitope shielding of the antigenic proteinusing high affinity IgM, IgD, IgA or IgG antibodies directed to the sameepitopes as the patient's IgE antibodies as described by Leroy et al[20]. Such antibodies may also bind close to the IgE specific epitopesthus preventing binding of the IgE antibodies by sterical hindrance.

Allergens are generally defined as antigens to which atopic patientsrespond with IgE antibody responses subsequently leading to allergicreactions. Antigens used in the molecule or the molecule complex of theinvention can be environmental allergens (e.g. house dust mite, birchpollen, grass pollen, cat antigens, cockroach antigens), or foodallergens (e.g. cow milk, peanut, shrimp, soya), or a combination ofboth. Also non relevant antigens such as HSA can be part of the moleculeor molecule complex according to the invention. The antigen can be acomplete allergen, exemplary an allergen for which patients with atopicdermatitis, allergic asthma, allergic rhinitis or allergicconjunctivitis are allergic. Preferably the allergen use in the moleculeor molecule complex according to the invention does not bind to IgE fromthe patient in need of treatment.

The antigens and/or epitopes used in the invention can be from naturalsources or be produced by recombinant technology or be producedsynthetically. Antigens and/or epitopes of the invention may containligand structures which facilitate incorporation of antigens and/orepitopes into molecule complexes of the invention via ligand/receptorinteractions or antibody binding. Antigens and/or epitopes of theinvention may contain chemical groups which facilitate covalent linkageof the antigens and/or epitopes to the CD32- and/or TLR9-bindingstructures of the molecule of the invention.

In one embodiment of the invention the antigens and epitopes of themolecule or molecule complex of the invention may be covalently linkedto the CD32 binding structure and/or to the TLR9 binding structure.

In one embodiment antigens and/or epitopes may also be linked by aligand/receptor interaction such as biotin and avidin to the molecule ormolecule complex of the invention. For example, the antigens or epitopesto be used in the molecule of the invention may be produced with biotinor a biotin mimetic attached to it. The CD32 binding structure and/orthe TLR9 binding structure may be produced with avidin or anotherbiotin-specific ligand attached to it. After mixing of these moleculeswith the different attachments, a stable molecule complex is formedaccording to the invention. Alternatively, an antibody/antigen bindingcan be used to form a molecule complex of the invention. High affinityinteractions are preferred for these embodiments (e.g. high affinityanti-digoxigenin antibody and digoxigenin labeled antigens and/orepitopes).

In one embodiment of the invention the antigens and/or epitopes aregenetically fused to the CD32-binding structure and/or to theTLR9-binding structure.

If the molecule of the invention is a fusion protein, the antigen ispreferably produced from at least one T-cell epitope-containingDNA-subsequence of an allergen. The T cell epitopes can alternatively befrom one or more related and/or unrelated allergens.

Preferably, the T cell epitopes comprise a new protein, which is not assuch a naturally existing protein and therefore is not recognized byexisting IgE or IgG antibodies in the patient. Therefore, instead ofselecting short T cell epitopes which are cut apart and fused togetheragain in a different order, one could also select a larger stretch of Tcell epitopes (>28 AA) which are still in their natural order but whichhave been previously selected not to bind to allergen specific IgE [21].

In principle all known antigens can be used for incorporation into themolecule or molecule complex of the invention to which allergic patientsrespond with IgE mediated hypersensitivity reactions. The most commonenvironmental allergens in the developed countries are: house dust mite,birch pollen, grass pollen, cat, and cockroach. Each of these allergenshas one or more “major allergens” (e.g. house dust mite: majorallergen=Der P1; Der F1, birch pollen: major allergen=Bet V1). However,complete antigens, though possible, are not necessary, because themolecule or molecule complex should only induce T cell responses, and Tcells respond to small (ca. 12-28 amino acid long) peptides presented inMHC Class Il molecules. The selection of T cell epitopes should bedesigned in such a way that expression on HLA class Il molecules ofpossibly all patients is guaranteed. Some HLA class Il molecules aremore frequently expressed than others. A good example for such a HLAclass Il molecule with wide expression is HLA DPw4, which is expressedon approximately 78% of the Caucasian population [22]. Therefore aselection of T cell epitopes could be included in the molecule ormolecule complex for each allergen, thus reducing the size and molecularweight of the complex. If overlapping cross-reactive epitopes betweenallergens from different genetically related organisms, such asDermatophagoides pteronyssinus (Der P1) and Dermatophagoides farinae,(Der F1), are present, they are preferred.

To allow for correct antigen processing, DNA coding for stretchesslightly longer than the actual T cell epitope should be included in themolecule or molecule complex and/or the epitopes can be separated fromeach other by introducing stretches of spacer DNA preferably containing(hydrophobic) epitopes recognized by major protein processing enzymes inantigen presenting cells such the asparagine-specific endopeptidase(AEP) or cathepsin S, cathepsin D or cathepsin L [23].

For fusion to the genes coding for the binding structures specific forTLR9 and CD32, preferably short DNA sequences of major allergens areused such as house dust mite major allergen I (Der P1, Der F1), housedust mite major allergen Il (Der P2, Der F2), or birch pollen allergen(Bet V1). These short DNA sequences contain the genetic code for one ormore T cell epitopes, which after processing, appear on the surface ofantigen presenting cells and therefore induce an immune response in theresponding allergen specific T cells. Not only T cell epitopes from DerP1 and Der P2 but also Der P3, Der P4, Der P5, Der P6, Der P7 etc. andDer F3, Der F4, Der F5, Der F6, Der F7 etc. can be used in a molecule ormolecule complex of the invention. T cell epitopes from these allergensmay be selected by classical epitope mapping using T cell clones [24] orby using modern HLA Class Il predicting software such as the Tepitopeprogram [25; 26]. For the molecule or molecule complexes, which can beformulated as vaccine, it is not necessary to combine T cell epitopesfrom a single allergen source only; to the contrary it is preferred toinclude as many T cell epitopes derived from different allergen sourcesproduced by one or many different species, e.g. a combination ofallergens from house dust mites and of allergens from grass pollen, catsand/or birch pollen.

As an example for Der P1 the majority of the T cell epitopes can befound in the following sequences 101-143 of the mature protein in aminoacid 1 letter code (SEQ ID No 4):

QSCRRPNAQ RFGISNYCQI YPPNANKIRE ALAQPQRYCR HYWT101       110        120        131       140 143

Especially the amino acid sequence 101-131 contains at least 3 T cellepitopes24, which bind to a number of HLA class Il molecules in aminoacid 1 letter code (SEQ ID No 5):

QSCRRPNAQ RFGISNYCQI YPPNANKIRE AL 101       110        120         131

The sequence 107-119 contains an important T cell epitope that binds toHLA DPw4 as well as HLA DPw5 24. These HLA Class Il molecules areexpressed by the majority of the population. The epitope in amino acid 1letter code (SEQ ID No 6):

NAQ RFGISNYCQI 107 110      119

Other important T cell epitopes which in addition are shared between DerP1 and Der F1 are found in the sequences 20-44 and 203-226 of the matureprotein in amino acid 1 letter code:

(SEQ ID No 7) RTVTPIRMQG GCGSCWAFSG VAATE 20         30         40  44and (SEQ ID No 8) YDGRTII QRDNGYQPNY HAVNIVGY203     210        220     227

Examples of T cells epitopes shared between Der P2 and Der F2 are foundin the sequence 26-44, 89-107 and 102-123

(SEQ ID No 9) PCII HRGKPFQLEA VFEAN 26   30         40  44(SEQ ID No 10) K YTWNVPKIAP KSENVVVT 89           100    107(SEQ ID No 11) ENVVVTVK VMGDDGVLAC AIAT 102      110        123 127

From the above mentioned T cell epitopes of Der P1/F1 and Der P2/F2 onecan design several functional molecule or molecule complexes, e.g.: Bytaking from Der P1 the following sequences:

(Sequence A, SEQ ID No 12) QSCRRPNAQ RFGISNYCQI YPP101       110        120 (Sequence B, SEQ ID No 13) CQI YPPNANKIRE AL117 120        130 (Sequence C, SEQ ID No 14) IRE ALAQPRYCR HYWT127 130       140 143 (Sequence D, SEQ ID No 7)RTVTPIRMQG GCGSCWAFSG VAATE 20         30         40  44(Sequence 3, SEQ ID No 8) YDGRTII QRDNGYQPNY HAVNIVGY203     210        220     227 And from Der P 2(Sequence F, SEQ ID No 9) PCII HRGKPFQLEA VFEAN 26   30         40   44(Sequence G, SEQ ID No 10) K YTWNVPKIAP KSENVVVT 89           100    107(Sequence H, SEQ ID No 11) ENVVVTVK VMGDDGVLAC AIAT102      110        120 123

One can design a cDNA with the order B,A,E,H,G,C,F,D or H,A,D,C,F,G,E,B,but any possible combination of the selected sequences will do. Thepreferred order of the epitopes will largely be determined on the basisof expression efficiency of the complete recombinant molecule. Alsoduplications of sequences are allowed e.g. B,B,A,E,E,G,C,G,F,A,D etc.The T cell epitope part may of course also contain the genetic codes forshorter peptides or longer peptides for more and for fewer peptides, aslong as one or more T cell epitopes from one or more differentallergens/antigens are included.

Epitopes from other allergens such as Bet V1, Lol P1, FeL d1 withsimilar characteristics will be preferred for inclusion in the moleculeor molecule complex according to the invention.

The invention also concerns a method of treating diseases, especiallyallergies, which comprises administering to a subject in need of suchtreatment a prophylactically or therapeutically effective amount of amolecule or molecule complex according to the invention for use as apharmaceutical, especially as an agent against allergies.

The molecule or molecule complex may be admixed with conventionalpharmaceutically acceptable diluents and carriers and, optionally, otherexcipients and administered parenterally intravenously or enterally,e.g. intramuscularly and subcutaneously. The concentrations of themolecule or molecule complex will, of course, vary depending, forexample, on the compound employed, the treatment desired and the natureof the form.

For different indications the appropriate doses will, of course, varydepending upon, for example, the molecule or molecule complex used, thehost, the mode of application and the intended indication. However, ingeneral, satisfactory results are indicated to be obtained with 1 to 4vaccinations in 1-2 years, but if necessary repeated additionalvaccination can be done. It is indicated that for these treatments themolecule or molecule complex of the invention may be administered in 2-4doses and with an application schedule similar as conventionallyemployed.

It further concerns a molecule or molecule complex according to theinvention for use as a pharmaceutical, particularly for use in thetreatment and prophylaxis of allergies.

The pharmaceutical composition prepared according to the presentinvention for use as vaccine formulation can (but does not have to)contain at least one adjuvant commonly used in the formulation ofvaccines apart from the molecule or molecule complex. It is possible toenhance the immune response by such adjuvants. As examples of adjuvants,however not being limited to these, the following can be listed:aluminum hydroxide (Alu gel), QS-21, Enhanzyn, derivatives oflipopolysaccharides, Bacillus Calmette Guerin (BCG), liposomepreparations, formulations with additional antigens against which theimmune system has already produced a strong immune response, such as forexample tetanus toxoid, Pseudomonas exotoxin, or constituents ofinfluenza viruses, optionally in a liposome preparation, biologicaladjuvants such as Granulocyte Macrophage Stimulating Factor (GM-CSF),interleukin 2 (IL-2) or gamma interferon (IFNγ). Aluminum hydroxide isthe most preferred vaccine adjuvant.

Summary of a Possible Mode of Action of the Fusion Protein According tothe Invention:

The fusion protein according to the present invention, can be formulatedin any of the available acceptable pharmaceutical formulations, but ispreferably formulated as a vaccine. The aCD32 binding portion of thefusion protein according to the invention selects the relevant cells.Triggering of CD32 on these cells will actively induce internalizationof the receptor plus the attached fusion protein and by doing sofacilitates the interaction of the TLR9 binding portion of the fusionprotein with the TLR9, which is expressed within the cytoplasm of therelevant antigen presenting cells [10; 11].

As a consequence of the CD32 mediated internalization, the subsequentprocessing and presentation of the selected T cell epitopes on MHC ClassIl molecules, combined with the specific activation of cytoplasmic TRL9in the antigen presenting cells, allergen specific T cells will be(re-)programmed to become Th1 memory cells. These allergen specific Th1memory cells at a later time point will induce allergen specific IgGproduction when encountering the same epitopes derived from the naturalallergens presented by naturally exposed allergen specific B cells.These TM cells thus are necessary for rebalancing the immune system fromIgE to IgG dominated antibody production.

EXAMPLES

The following examples shall explain the present invention in moredetail, without, however, restricting it.

Example 1 Panning of the Human CL-Phage Library on a TLR-9 Peptide e.g.Sequence 216-240 of the Mature Protein TLR9 (SEQ ID No 3) in Amino Acid1 Letter Code

ANLT ALRVLDVGGN CRRCDHAPNP C 216  220        230        240

3 panning rounds shall be performed according to standard protocols.Briefly, the following method can be applied. Maxisorp 96-well plates(Nunc) are coated with the (synthetic) peptide representing part of thesequence of the TLR-9. For coating the peptides in the wells, 200 μl ofthe following solution are added per well: 0.1 M

Na-carbonate buffer, pH 9.6, with the following concentrations ofdissolved peptide:

1st panning round: 1 mg/ml TLR-9 peptide

2nd panning round: 500 μg/ml TLR-9 peptide

3rd panning round: 100 μg/ml TLR-9 peptide

Incubation is for 1 hour at 37° C., followed by blocking with 2% drymilk (M-PBS) with 200 μl per well for 1 hour at room temperature. Thesurface display phage library is then allowed to react with the boundpeptide by adding 100 μl phage suspension and 100 μl 4% dry milk(M-PBS), followed by incubation for 45 minutes with shaking and for 90minutes without shaking at room temperature. Unbound phage particles arewashed away as follows. After the 1st panning round: 10×300 μl T-PBS,5×300 μl PBS; after the 2nd panning round: 15×300 μl T-PBS, 10×300 μlPBS; after the 3rd panning round: 20×300 μl T-PBS, 20×300 μl PBS.Elution of bound phage particles is performed by adding 200 μl per wellof 0.1 M glycine, pH 2.2, and incubation with shaking for 30 minutes atroom temperature. Subsequently, the phage suspension is neutralized byaddition of 60 μl 2M Tris-Base, followed by infection into E. coli TG1cells by mixing 10 ml exponentially growing culture with 0.5 ml elutedphage and incubation for 30 minutes at 37° C. Finally, infected bacteriaare plated on TYE medium with 1% glucose and 100 μg/ml Ampicillin, andincubated at 30° C. overnight.

Example 2 Cloning of Selected Clones of Human CL Mutants SelectedAgainst TLR-9 for Soluble Expression

Phagemid DNA from the phage selected through the 3 panning rounds isisolated with a midi-prep. DNA encoding mutated CL-regions isbatch-amplified by PCR and cloned Nco1-Not1 into the vectorpNOTBAD/Myc-His, which is the E. coli expression vector pBAD/Myc-His(Invitrogen) with an inserted Not1 restriction site to facilitatecloning. Ligated constructs are transformed into E. coli LMG194 cells(Invitrogen) with electroporation, and grown at 30° C. on TYE mediumwith 1% glucose and ampicillin overnight. Selected clones are inoculatedinto 200 μl 2×YT medium with ampicillin, grown overnight at 30° C., andinduced by adding L-arabinose to an end concentration of 0.1%. Afterexpression at 16° C. overnight, the cells are harvested bycentrifugation and treated with 100 μl Na-borate buffer, pH 8.0, at 4°C. overnight for preparation of periplasmic extracts. 50 μl of theperiplasmic extracts were used in ELISA (see below).

Example 3: ELISA of Human CL Mutants Selected Against TLR-9

Selected clones are assayed for specific binding to the TLR-9 peptide byELISA.

Coating: Microtiter plate (NUNC, Maxisorp), 100 μl per well, 20 μg TLR-9peptide/ml 0.1 M Na-carbonate buffer, pH 9.6, 1 h at 37° C.

Wash: 3×200 μl PBS

Blocking: 1% BSA-PBS, 1 h at RT

Wash: 3×200 μl PBS

Periplasmic extract binding: 50 μl periplasmic extract

50 μl 2% BSA-PBS, at room temperature overnight

Wash: 3×200 μl PBS

1st antibody: anti-His4 (Qiagen), 1:1000 in 1% BSA-PBS, 90 min at RT,100 μl per well

Wash: 3×200 μl PBS

2nd antibody: goat anti mouse*HRP (SIGMA), 1:1000 in 1% BSA-PBS, 90 minat RT, 100 μl per well

Wash: 3×200 μl PBS

Detection: 3 mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5, 0.4 μl30% H2O2

Stopping: 100 ml 3M H2SO4

Absorbance read: 492/620 nm

Clones that give a high signal in this first, preliminary ELISA arecultured in a 20-ml volume at the same conditions as described above.Their periplasmic extracts are isolated in 1/20 of the culture volume asdescribed above and tested with ELISA (as described above) forconfirmation.

Example 4: Cloning of the Anti-CD32 Variable Domains from HB-217

mRNA is isolated from the cell line HB-217 (ATCC, antiCD32 antibodyIV.3) and is used to prepare cDNA according to established routineprotocols. The cDNA is further used as a template to amplify the regionsof the genes coding for the of the light and the heavy chain of the Fabfragment of antibody IV.3 respectively. Upstream PCR primers, whichprime from the 5′ end of the variable regions, used for thisamplification are derived from the published sequences of mouse variableregions (IMGT, the international ImMunoGeneTics information System®).Degenerate primers and/or mixtures of different primers are used asupstream primers. Downstream primers are designed such as to prime fromthe 31 end of the CL or the CH 1 domains respectively.

In a next step, the CL domain of the antibody IV.3 is removed andreplaced by a selected CL domain modified by SMID technology which hasbinding affinity to TLR9, and which is selected as described above inexamples 1-3. For this replacement, overlapping PCR can be usedaccording to standard protocols. Alternatively, for joining VL to theSMID modified CL a uniquely cutting restriction site can be used whichis either naturally occurring in the sequence or which is artificiallyintroduced by site directed mutagenesis (as a silent mutation which doesnot change the amino acid sequence). For example, a BstAPI site can begenerated in the hinge region between VL and CL by changing the sequencefrom:

(SEQ ID No 65)  K  R  A  D  A  A  P  T  V  S  I  F (SEQ ID No 66)AAACGGGCTGATGCTGCACCAACTGTATCCATCTTC to: (SEQ ID No 65) K  R  A  D  A  A  P  T  V  S  I  F (SEQ ID No 15) AAACGG GCAGATGCTGCACCAACTGTATCCATCTTC

the newly created BstAPI site is highlighted in the above sequence. Thenew sequence is introduced in the coding regions by amplifying the VLpart and the CL part respectively with appropriately designed PCRprimers, cutting the PCR products with BstAPI, ligating them, andamplifying the complete resulting ligation product with PCR primers asused initially for amplifying the original light chain part of the Fabfragment.

For expression of the modified Fab fragment, the genes coding for theheavy and the light chains are subsequently cloned in appropriateexpression vectors, or together in one expression vector which allowsthe expression of two independent genes. As an expression system,bacteria, yeast, animal cells or any other suitable expression systemcan be used. For this example here, expression from one vector in themethylotrophic yeast Pichia pastoris will be shown.

The light chain part of the modified PCR fragment is cloned EcoRI/Kpn1in the Pichia pastoris expression vector pPICZalphaA in the correctreading frame such as to fuse it functionally with the alpha-factorsecretion signal sequence provided by the vector. Similarly, the heavychain part of the Fab fragment is cloned in pPICZalphaA. In order toprepare the inserts for this cloning procedure, appropriately designedPCR primers are used which attach the needed restriction sites to thegenes. At the 31 ends of both coding regions, a stop codon has to beinserted and provided by the PCR primers as well. The light chainexpression cassette is then cut out from the vector with restrictionenzymes BgIII and BamHI, and the ends of the DNA are made blunt bytreatment with Klenow fragment of DNA polymerase. The vector containingthe inserted heavy chain part of the Fab is opened by a partial digestwith restriction enzyme BgIII, the DNA is made blunt by treatment withKlenow fragment of DNA polymerase, and the expression cassette codingfor the light chain part is inserted. The partial digest of the heavychain vector is necessary since the inserted heavy chain gene contains aBgIII site. For screening of the final construct, care has to be takenthat this internal BgIII site has remained intact. The final constructhas one Pme1 site which is used for linearizing the construct prior totransformation into Pichia pastoris. This linearization is advantageousfor efficient integration of the expression vector in the host genome byhomologous recombination. Pichia pastoris is transformed with thelinearized expression vector using electroporation, transformed clonesare selected with the antibiotic Zeocin for which the vector confersresistance, and supernatants of randomly picked clones are screened forexpression of the Fab construct after induction of expression withmethanol. For screening, e.g. a Fab-specific ELISA can be used.Production of the recombinant protein is achieved by culturing thetransformed selected Pichia clone in a larger scale, preferable in shakeflasks or in a fermenter, inducing expression by addition of methanoland purifying the recombinant protein by a chromatographic method. Forthese latter steps, routine protocols are used.

Example 5: Cloning of the Der P1I/F1 and Der P2/F2 derived T CellEpitopes

-   -   The combination of the selected T cell epitopes formed by        sequences B,A,E,H,G,C,F,D looks as follows (SEQ ID No 16):

CQIYPPNANKIREAL  QSCRRPNAQRFGISNYCQIYPP  YDGRTIIQRDNGYQPNYHAVNI    (Seq: B)            (Seq. A)                 (Seq. E) VGY ENVVVTVKVMGDDGVLACAIAT  KYTWNVPKIAPKSENVVVT  IREALAQPQRYCRH            (Seq. H)               (Seq. G)       (Seg. C) YWT PCIIHRGKPFQLEAVFEAN  RTVTPIRMQGGCGSCWAFSGVAATE          (Seq. F)                 (Seq. D)

In order to construct a synthetic gene coding for this amino acidsequence, in silico reverse translation can be used. Computer programsare available for this purpose, such as e.g. DNAWORKS, in order to clonethe synthetic gene coding for the epitopes in frame with the gene codingfor the heavy chain part of the framework, two restriction sites areselected which cut neither on this coding region nor on the vectorpPICZalphaA. For example, AccIII and SpeI can be used for this purpose.These two restriction sites are attached to the gene coding for theheavy chain part of the Fab by using appropriately designed PCR primersfor the cloning procedure as described above. Furthermore, care has tobe taken not to have a stop codon at the end of the coding region of theheavy chain part of the Fab, as the stop codon will be provided at the3′ end of the synthetic gene coding for the epitopes. Again, thisconstruct with the two additional restriction sites located at its 3′end is cloned EcoRI/Kpn1 in the Pichia pastoris expression vectorpPICZalphaA. The construct is then opened with the restriction enzymesAccIII and SpeI and the insert coding the epitopes in inserted. Thisinsert is generated as follows:

The chosen amino acid sequence:

(SEQ ID No 16) CQIYPPNANKIREAL  QSCRRPNAQRFGISNYCQIYPP  YDGRTIIQRDNGYQPNYHAVNIVGY  ENVVVTVKVMGDDGVLACAIAT  KYTWNVPKIAPK SENVVVT IREALAQPQRYCRHYWT  PCIIHRGKPFQLEAVFEAN  RTVT PIRMQGGCGSCWAFSGVAATE

together with the chosen restriction sites, in this example AccIII atthe 5′ end and SpeI at the 3′ end are used as input in the publiclyavailable computer program DNAWORKS. In addition, a stop codon is addedbetween the end of the epitope sequence and the SpeI site.

The parameters which the program uses for designing the oligonucleotidesare left at the proposed standard values, and the program is instructedto avoid the sequences of the restriction sites which are necessary forthe cloning and transformation steps, such as AccIII, SpeI and PmeI.

AccIII: tccgga SpeI: actagt PmeI: gtttaaac

DNAWORKS generates a set of oligonucleotides which are overlapping andwhich represent both strands of the desired coding regions.

For example, the following set of 24 oligonucleotides is generated, fromwhich the synthetic gene coding for the allergen epitopes is generated:

(SEQ ID No 17)  1 TCCGGATGCCAAATTTACCCGCCAAACG 28 (SEQ ID No 18)  2AGCCTCTCTGATCTTGTTCGCGTTTGGCGGGTAAATTTGG 40 (SEQ ID No 19)  3CGAACAAGATCAGAGAGGCTTTGCAATCTTGCAGGAGGCC 40 (SEQ ID No 20)  4TATGCCGAATCTCTGCGCATTGGGCCTCCTGCAAGATTGC 40 (SEQ ID No 21)  5GCGCAGAGATTCGGCATATCCAACTACTGCCAGATCTACC 40 (SEQ ID No 22)  6GTACGCCCATCGTATGGGGGGTAGATCTGGCAGTAGTTGG 40 (SEQ ID No 23)  7CCCATACGATGGGCGTACAATCATACAGCGTGATAACGGC 40 (SEQ ID No 24)  8GCGTGGTAGTTAGGCTGATAGCCGTTATCACGCTGTATGA 40 (SEQ ID No 25)  9TATCAGCCTAACTACCACGCCGTGAACATCGTCGGCTACG 40 (SEQ ID No 26) 10TCACAGTAACCACGACATTCTCGTAGCCGACGATGTTCAC 40 (SEQ ID No 27) 11AGAATGTCGTGGTTACTGTGAAGGTAATGGGCGATGACGG 40 (SEQ ID No 28) 12AGCTATGGCGCAAGCTAGAACCCCGTCATCGCCCATTACC 40 (SEQ ID No 29) 13TCTAGCTTGCGCCATAGCTACCAAGTACACTTGGAACGTA 40 (SEQ ID No 30) 14TTTTCGGCGCAATTTTGGGTACGTTCCAAGTGTACTTGGT 40 (SEQ ID No 31) 15CCCAAAATTGCGCCGAAAAGTGAAAACGTCGTAGTGACCA 40 (SEQ ID No 32) 16TGAGCCAATGCCTCCCTTATGGTCACTACGACGTTTTCAC 40 (SEQ ID No 33) 17AGGGAGGCATTGGCTCAACCTCAAAGATACTGCAGACACT 40 (SEQ ID No 34) 18TTATGCAGGGCGTCCAGTAGTGTCTGCAGTATCTTTGAGG 40 (SEQ ID No 35) 19ACTGGACGCCCTGCATAATCCACCGTGGTAAACCCTTTCA 40 (SEQ ID No 36) 20CTTCGAACACTGCCTCAAGTTGAAAGGGTTTACCACGGTG 40 (SEQ ID No 37) 21ACTTGAGGCAGTGTTCGAAGCTAACAGGACGGTAACGCCA 40 (SEQ ID No 38) 22CCGCACCCACCTTGCATACGAATTGGCGTTACCGTCCTGT 40 (SEQ ID No 39) 23TGCAAGGTGGGTGCGGGTCTTGTTGGGCTTTTTCTGGTGT 40 (SEQ ID No 40) 24ACTAGTTTATTCAGTAGCAGCCACACCAGAAAAAGCCCAACA 42

These 24 oligonucleotides are dissolved, mixed together, boiled forseveral minutes and then cooled down to room temperature slowly to allowannealing. In a subsequent PCR steps using large amounts of the twobordering primers (primers #1 and #24), the annealed gene is amplified,the PCR product is then cleaved with the chosen restriction enzymes(AccIII and SpeI in this example), and cloned into the expression vectoras described above, which contains as an insert the gene coding for theheavy chain part of the modified Fab. Preparation of the finalexpression vector containing both chains, transformation of Pichiapastoris, selection of clones and screening for producing clones is doneas described above. Expression and purification of the recombinantprotein is performed by following standard protocols.

Example 6: Fusion of VH and VL of the Anti-CD32 Antibody IV.3 Fusionwith Anti-TLR9CH3 Domains (SMIDS)

All molecular modeling was done with Swiss-PdbViewer 3.7.

As a homology model for a mouse Fab fragment, the structure file2BRR.pdb from the Protein Data Bank is used, and 1OQO.pdb is used as asource for the structure of a human IgG CH3 domain.

Molecular models of VH and VL of the IV.3 antibody are made with the“first approach mode” of the Swissmodel system using the amino acidsequences of VH and VL respectively.

Using the “magic fit” function of the Swiss-PdbViewer, two copies of theCH3 domain structure from 1OQO.pdb are fitted onto the CH1 and the CLdomain respectively of 2BRR.pdb. Subsequently, the molecular models ofthe IV.3 VH and VL respectively are fitted (again using “magic fit”)onto VH and VL of 2BRR.pdb.

For construction of a Fab-like protein in which CH 1 and CL are bothreplaced by a CH3 domain, it is necessary to decide at which point thesequence of VH should be ended and connected to the sequence of CH3, andat which point the sequence of VL should be ended and connected to thesequence of CH3. For both constructs, a point is chosen at which themain chain of the superimposed structures and models (see above) showsan optimal overlap.

For the light chain, it was found that the sequence up to Ala114(numbering from 2BRR.pdb) will be used an connected to Pro343 (numberingfrom 1OQO.pdb) of the CH3 domain. The point of connection between thesetwo sequences therefore reads as follows (VL part is underlined): - - -Lys112-Arg113-Ala114-Pro343-Arg344-Glu345- -

In order to allow joining of the two coding sequences using restrictionenzyme sites and DNA ligation, the sequence near the point of connectionis changed by silent mutation to introduce a unique Xho1 site (ctcgag,underlined) as follows:

(SEQ ID No 41)  K  R  A  P  R  E (SEQ ID No 42) AAACGGGCTC CTCGAG AA

For later insertion of the allergen epitopes, an AscI site (ggcgcgcc) isintroduced just before the stop codon of the construct plus an extrabase for maintenance of the reading frame:

ggg cgc gcc Gly Arg Ala

Furthermore, for cloning into the expression vector pPICZalphaA (Pichiapastoris expression system, Invitrogen), an EcoRI site (gaattc) is addedto the 5′-end (N-terminus) and a Kpn1 site (ggtacc) to the 3′-end(C-terminus) of the construct.

The CH3 domain to be fused to VH and VL respectively selected as part ofthe construct can be a wild type human IgG CH3 domain which can serve asa negative control, or a CH3 domain previously engineered by SMIDtechnology and selected to bind specifically to TLR9. In this examplehere, the sequence of clone A23, which binds specifically to TLR9 andwhich was described in the patent application PCT/EP2006/050059 is fusedto both, VH and VL.

Therefore, the complete sequence of the VL-CH3 fusion protein has thefollowing amino acid sequence (VL part is underlined), (SEQ ID No 43):

DIVMTQAAPS VPVTPGESVS ISCRSSKSLL HTNGNTYLHWFLQRPGQSPQ LLIYRMSVLA SGVPDRFSGS GSGTAFTLSISRVEAEDVGV FYCMQHLEYP LTFGAGTKLE LKRAPREPQVYTLPPSRDEL GIAQVSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLYS KLTVLGRRWT LGNVFSCSVM HEALHNHYTQ KSLSLSPGK&

Nucleic acid sequence of the VL-CH3 fusion protein (restriction sitesare underlined), (SEQ ID No 44):

gaattcGACA TTGTGATGAC CCAGGCTGCA CCCTCTGTACCTGTCACTCC TGGAGAGTCA GTATCCATCT CCTGCAGGTCTAGTAAGAGT CTCCTGCATA CTAATGGCAA CACTTACTTGCATTGGTTCC TACAGAGGCC AGGCCAGTCT CCTCAGCTCCTGATATATCG GATGTCCGTC CTTGCCTCAG GAGTCCCAGACAGGTTCAGT GGCAGTGGGT CAGGAACTGC TTTCACACTGAGCATCAGTA GAGTGGAGGC TGAGGATGTG GGTGTTTTTTACTGTATGCA ACATCTAGAA TATCCGCTCA CGTTCGGTGCTGGGACCAAG CTGGAACTGA AACGGGCTCC TCGAGAACCACAGGTGTACA CCCTGCCCCC ATCCCGGGAC GAGCTCGGCATCGCGCAAGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTATCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAACGGGCAGCCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC TTTCTTCCTC TACAGCAAGC TTACCGTGTTGGGCCGCAGG TGGACCCTGG GGAACGTCTT CTCATGCTCCGTGATGCATG AGGCTCTGCA CAACCACTAC ACACAGAAGAGCCTCTCCCT GTCTCCGGGT AAATGAgggc gcgccggtac c

For the heavy chain, it was found that the sequence up to ThM 23(numbering from 2BRR.pdb) should be used an connected to Arg344(numbering from 1OQO.pdb) of the CH3 domain. The point of connectionbetween these two sequences therefore reads as follows (VH part isunderlined): - - - Ala121-Lys122-Thr123-Arg344-Glu345-Pro346 - - -

In order to allow joining of the two coding sequences using restrictionenzyme sites and DNA ligation, the sequence near the point of connectionwas changed by silent mutation to introduce a unique Xho1 site (ctcgag,underlined) as follows:

(SEQ ID No 45)  A  K  T  R  E  P (SEQ ID No 46) GCCAAAACTCGAGAACCA

Furthermore, for cloning into the expression vector pPICZalphaA (Pichiapastoris expression system, Invitrogen), an EcoRI site (gaattc) is addedto the 5′-end (N-terminus) and an Xba1 site (tctaga) to the 3′-end(C-terminus) of the construct. No stop codon is added to this sequenceand the Xba1 site is placed in the correct reading frame so as to fusethe construct to the Hexa-His-tag provided by the vector for laterpurification of the protein using immobilized metal affinitychromatography.

Therefore, the complete sequence of the VH-CH3 fusion protein has thefollowing amino acid sequence (VH part is underlined), (SEQ ID No 47):

EVQLQQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQAPGKGLKWMGW LNTYTGESIY PDDFKGRFAF SSETSASTAYLQINNLKNED MATYFCARGD YGYGGPLDYW GQGTSVTVSSAKTREPQVYT LPPSRDELGI AQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVLGRRWTLGNVFSCSVMHE ALHNHYTQKS LSLSPGKSLE QKLISEEDLN SAVDHHHHHH&

Nucleic acid sequence of the VH-CH3 fusion protein (restriction sitesare underlined), (SEQ ID No 48):

GAATTCGAGG TTCAGCTTCA GCAGTCTGGA CCTGAGCTGAAGAAGCCTGG AGAGACAGTC AAGATCTCCT GCAAGGCTTCTGGGTATACC TTCACAAACT ATGGAATGAA CTGGGTGAAGCAGGCTCCAG GAAAGGGTTT AAAGTGGATG GGCTGGTTAAACACCTACAC TGGAGAGTCA ATATATCCTG ATGACTTCAAGGGACGGTTT GCCTTCTCTT CGGAAACCTC TGCCAGCACTGCCTATTTGC AGATCAACAA CCTCAAAAAT GAGGACATGGCTACATATTT CTGTGCAAGA GGGGACTATG GTTACGACGACCCTTTGGAC TACTGGGGTC AAGGAACCTC AGTCACCGTCTCCTCAGCCA AAACTCGAGA ACCACAGGTG TACACCCTGCCCCCATCCCG GGACGAGCTC GGCATCGCGC AAGTCAGCCT GACCTGCCTGGTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGGAGAGCAACGG GCAGCCGGAG AACAACTACA AGACCACGCCTCCCGTGCTG GACTCCGACG GCTCTTTCTT CCTCTACAGCAAGCTTACCG TGTTGGGCCG CAGGTGGACC CTGGGGAACGTCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCACTACACACAG AAGAGCCTCT CCCTGTCTCC GGGTAAATCTCTAGAACAAA AACTCATCTC AGAAGAGGAT CTGAATAGCGCCGTCGACCA TCATCATCAT CATCATTGA

Detailed Cloning Plan

Heavy Chain:

The VH region of antibody IV.3 is PCR-amplified with primers 4.3HupEcoand 4.3HdownXho, and subsequently digested with EcoRI and Xho1. The CH3SMID-engineered clone A23 is PCR-amplified with primers CH3upXhoA andCH3XBA2 and subsequently digested with Xho1 and Xba1. The VH sequenceand the CH3 sequence are ligated together via the Xho1 site and thenligated into pPICZalphaA (Invitrogen), which was previously digestedwith EcoRI and Xba1. The resulting vector is named pPICHA23.

Primer List:

(SEQ ID No 49) 4.3HUPECO cagagaattc gaggttcagc ttcagcagtc (SEQ ID No 50)4.3HDOWNXHO gatgctcgag ttttggctga ggagacggtg (SEQ ID No 51) CH3UPXHOAaaaactcgag aaccacaggt gtacaccctg cc (SEQ ID No 52) CH3XBA2actgatctag acctttaccc ggagacaggg agag

Light Chain:

The VL region of antibody IV.3 is PCR-amplified with primers 4.3LupEcoand 4.3LdownXho, and subsequently digested with EcoRI and Xho1. The CH3SMID-engineered clone A23 is PCR-amplified with primers CH3upXhoB andCH3StopKpn and subsequently digested with Xho1 and Kpn1. The VL sequenceand the CH3 sequence are ligated together via the Xho1 site and thenligated into pPICZalphaA (Invitrogen), which was previously digestedwith EcoRI and Kpn1. The resulting vector is named pPICLA23.

Primer List:

(SEQ ID No 53) 4.3LUPECO gatagaattc gacattgtga tgacccaggc tg(SEQ ID No 54) 4.3LDOWNXHO attactcgag gagcccgttt cagttccagc t(SEQ ID No 55) CH3UPXHOB gctcctcgag aaccacaggt gtacaccctg cc(SEQ ID No 56) CH3STOPKPN acgtggtacc tcaggcgcgc cctttacccggagacaggga gag

Combination of the Two Expression Cassettes in One Vector

The light chain cassette is cut out with BgIII (pos.1) and BamHI (pos.2319) from pPICLA23 (4235 bp), and the 2319 bp fragment is purified viapreparative gel electrophoresis. The 1916 bp fragment is discarded. Thevector pPICHA23 (4219 bp) is digested with BamHI, and the previouslypurified 2319 bp fragment from pPICLA23 is inserted. The resultingPichia pastoris expression vector, which carries two expressioncassettes, one for the VL-CH3 fusion protein and on for the VH-CH3fusion protein is screened so that both inserts that have same directionof transcription. The resulting vector pPICHLA23 (6537 bp) is thenlinearized before transformation into Pichia pastoris e.g. with BamHI orwith BssSI, transformed into Pichia pastoris by electroporation, andpositive transformants are selected with Zeocin. Several clones arescreened for expression of the recombinant protein. A clone is thenselected for large scale production, and the recombinant fusion proteinis purified by immobilized-metal-affinity chromatography using standardprocedures. All Pichia manipulation, culturing and expression is done byfollowing standard protocols (Invitrogen).

Insertion of allergen epitopes into the vector pPICHLA23 and expressionof the recombinant fusion protein

The sequence encoding the allergen epitopes as described in example 5 isinserted into the vector pPICHLA23 as follows:

The vector is digested with AscI (4174-4182) which leads to itslinearization. In this AscI site, the DNA sequence encoding the allergenepitopes is inserted. The sequence encoding the allergen epitopes isamplified with primers EpiTLRI and EpiTLR2 in order to attach AscI sitesto both ends of the sequence.

Primer List

(SEQ ID No 57) EpiTLR1 TAAAGGGCGC GCCTCCGGAT GCCAAATTTA CC(SEQ ID No 58) EpiTLR2 TACCTCAGGC GCGCCTTATT CAGTAGCAGC CACAC

The resulting PCR product is digested with AscI and ligated into thepreviously digested vector. The resulting vector is named pHLA23EP (7046bp). Pichia transformation, expression and purification of therecombinant fusion protein is performed as described above for theconstruct that has no epitopes inserted.

VL of Antibody IV.3

Amino Acid Sequence:

(SEQ ID No 59) DIVMTQAAPS VPVTPGESVS ISCRSSKSLL HTNGNTYLHWFLQRPGQSPQ LLIYRMSVLA SGVPDRFSGS GSGTAFTLSISRVEAEDVGV FYCMQHLEYP LTFGAGTKLE LKRA

Nucleic Acid Sequence:

(SEQ ID No 60) GACATTGTGA TGACCCAGGC TGCACCCTCT GTACCTGTCACTCCTGGAGA GTCAGTATCC ATCTCCTGCA GGTCTAGTAAGAGTCTCCTG CATACTAATG GCAACACTTA CTTGCATTGGTTCCTACAGA GGCCAGGCCA GTCTCCTCAG CTCCTGATATATCGGATGTC CGTCCTTGCC TCAGGAGTCC CAGACAGGTTCAGTGGCAGT GGGTCAGGAA CTGCTTTCAC ACTGAGCATCAGTAGAGTGG AGGCTGAGGA TGTGGGTGTT TTTTACTGTATGCAACATCT AGAATATCCG CTCACGTTCG GTGCTGGGAC CAAGCTGGAA CTGAAACGGG CT

VH of Antibody IV.3

Amino Acid Sequence:

(SEQ ID No 61) EVQLQQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQAPGKGLKWMGW LNTYTGESIY PDDFKGRFAF SSETSASTAYLQINNLKNED MATYFCARGD YGYDDPLDYW GQGTSVTVGS AKT

Nucleic Acid Sequence:

(SEQ ID No 62) GAGGTTCAGC TTCAGCAGTC TGGACCTGAG CTGAAGAAGCCTGGAGAGAC AGTCAAGATC TCCTGCAAGG CTTCTGGGTATACCTTCACA AACTATGGAA TGAACTGGGT GAAGCAGGCTCCAGGAAAGG GTTTAAAGTG GATGGGCTGG TTAAACACCTACACTGGAGA GTCAATATAT CCTGATGACT TCAAGGGACGGTTTGCCTTC TCTTCGGAAA CCTCTGCCAG CACTGCCTATTTGCAGATCA ACAACCTCAA AAATGAGGAC ATGGCTACATATTTCTGTGC AAGAGGGGAC TATGGTTACG ACGACCCTTTGGACTACTGG GGTCAAGGAA CCTCAGTCAC CGTCTCCTCA GCCAAAACA

Final Expression Vector pPICHCLA23.seq. (SEQ ID No 63) Containing TLR9and CD32 Binding Regions (6537 bp):

1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 61gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 121tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 181agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 241acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 301tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 361agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 421gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 481ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 541cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 601ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 661ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 721gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 781atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 841actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 901caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 961tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1021agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1081tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1141tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1201tgaagctgaa ttcgaggttc agcttcagca gtctggacct gagctgaaga agcctggaga 1261gacagtcaag atctcctgca aggcttctgg gtataccttc acaaactatg gaatgaactg 1321ggtgaagcag gctccaggaa agggtttaaa gtggatgggc tggttaaaca cctacactgg 1381agagtcaata tatcctgatg acttcaaggg acggtttgcc ttctcttcgg aaacctctgc 1441cagcactgcc tatttgcaga tcaacaacct caaaaatgag gacatggcta catatttctg 1501tgcaagaggg gactatggtt acgacgaccc tttggactac tggggtcaag gaacctcagt 1561caccgtctcc tcagccaaaa ctcgagaacc acaggtgtac accctgcccc catcccggga 1621tgagctgggc atcgcgcaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1681catcgccgtg gagtgggaga gcaacgggca gccggagaac aactacaaga ccacgcctcc 1741cgtgctggac tccgacggct ctttcttcct ctacagcaag cttaccgtgt tgggccgcag 1801gtggaccctg gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1861cacgcagaag agcctctccc tgtctccggg taaatctcta gaacaaaaac tcatctcaga 1921agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt gtagccttag 1981acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct 2041aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt 2101ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga 2161gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa 2221aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat 2281taagtgagac cttcgtttgt gcagatccaa catccaaaga cgaaaggttg aatgaaacct 2341ttttgccatc cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg 2401ggatacacta gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac 2461ccacttttgc catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc 2521aattccttct attaggctac taacaccatg actttattag cctgtctatc ctggcccccc 2581tggcgaggtt catgtttgtt tatttccgaa tgcaacaagc tccgcattac acccgaacat 2641cactccagat gagggctttc tgagtgtggg gtcaaatagt ttcatgttcc ccaaatggcc 2701caaaactgac agtttaaacg ctgtcttgga acctaatatg acaaaagcgt gatctcatcc 2761aagatgaact aagtttggtt cgttgaaatg ctaacggcca gttggtcaaa aagaaacttc 2821caaaagtcgg cataccgttt gtcttgtttg gtattgattg acgaatgctc aaaaataatc 2881tcattaatgc ttagcgcagt ctctctatcg cttctgaacc ccggtgcacc tgtgccgaaa 2941cgcaaatggg gaaacacccg ctttttggat gattatgcat tgtctccaca ttgtatgctt 3001ccaagattct ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt 3061ctaaccccta cttgacagca atatataaac agaaggaagc tgccctgtct taaacctttt 3121tttttatcat cattattagc ttactttcat aattgcgact ggttccaatt gacaagcttt 3181tgattttaac gacttttaac gacaacttga gaagatcaaa aaacaactaa ttattcgaaa 3241cgatgagatt tccttcaatt tttactgctg ttttattcgc agcatcctcc gcattagctg 3301ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa gctgtcatcg 3361gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc aacagcacaa 3421ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa gaagaagggg 3481tatctctcga gaaaagagag gctgaagctg aattcgacat tgtgatgacc caggctgcac 3541cctctgtacc tgtcactcct ggagagtcag tatccatctc ctgcaggtct agtaagagtc 3601tcctgcatac taatggcaac acttacttgc attggttcct acagaggcca ggccagtctc 3661ctcagctcct gatatatcgg atgtccgtcc ttgcctcagg agtcccagac aggttcagtg 3721gcagtgggtc aggaactgct ttcacactga gcatcagtag agtggaggct gaggatgtgg 3781gtgtttttta ctgtatgcaa catctagaat atccgctcac gttcggtgct gggaccaagc 3841tggaactgaa acgggctcct cgagaaccac aggtgtacac cctgccccca tcccgggatg 3901agctgggcat cgcgcaagtc agcctgacct gcctggtcaa aggcttctat cccagcgaca 3961tcgccgtgga gtgggagagc aacgggcagc cggagaacaa ctacaagacc acgcctcccg 4021tgctggactc cgacggctct ttcttcctct acagcaagct taccgtgttg ggccgcaggt 4081ggaccctggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac aaccactaca 4141cgcagaagag cctctccctg tctccgggta aagggcgcgc ctgaggtacc tcgagccgcg 4201gcggccgcca gctttctaga acaaaaactc atctcagaag aggatctgaa tagcgccgtc 4261gaccatcatc atcatcatca ttgagtttgt agccttagac atgactgttc ctcagttcaa 4321gttgggcact tacgagaaga ccggtcttgc tagattctaa tcaagaggat gtcagaatgc 4381catttgcctg agagatgcag gcttcatttt tgatactttt ttatttgtaa cctatatagt 4441ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga tcagcctatc 4501tcgcagctga tgaatatctt gtggtagggg tttgggaaaa tcattcgagt ttgatgtttt 4561tcttggtatt tcccactcct cttcagagta cagaagatta agtgagacct tcgtttgtgc 4621ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt 4681tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt 4741tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa 4801aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt 4861tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc cattgatatt 4921taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac 4981aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct aaggggcggt 5041gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac 5101taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga 5161gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc 5221gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg 5281ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg 5341tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc 5401gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc 5461gtggccgagg agcaggactg acacgtccga cggcggccca cgggtcccag gcctcggaga 5521tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg 5581ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt 5641ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt 5701tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga 5761gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca tgtgagcaaa 5821aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5881ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5941aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 6001gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 6061tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 6121tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 6181gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 6241cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 6301cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 6361agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 6421caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 6481ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagatc

Final Expression Vector pHLA23EP.seq (SEQ ID No 64) Containing TLR9 andCD32 Binding Regions and Epitope Sequence (See SEQ ID No 16) (7046 bp):

1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 61gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 121tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 181agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 241acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 301tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 361agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 421gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 481ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 541cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 601ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 661ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 721gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 781atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 841actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 901caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 961tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1021agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1081tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1141tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1201tgaagctgaa ttcgaggttc agcttcagca gtctggacct gagctgaaga agcctggaga 1261gacagtcaag atctcctgca aggcttctgg gtataccttc acaaactatg gaatgaactg 1321ggtgaagcag gctccaggaa agggtttaaa gtggatgggc tggttaaaca cctacactgg 1381agagtcaata tatcctgatg acttcaaggg acggtttgcc ttctcttcgg aaacctctgc 1441cagcactgcc tatttgcaga tcaacaacct caaaaatgag gacatggcta catatttctg 1501tgcaagaggg gactatggtt acgacgaccc tttggactac tggggtcaag gaacctcagt 1561caccgtctcc tcagccaaaa ctcgagaacc acaggtgtac accctgcccc catcccggga 1621tgagctgggc atcgcgcaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1681catcgccgtg gagtgggaga gcaacgggca gccggagaac aactacaaga ccacgcctcc 1741cgtgctggac tccgacggct ctttcttcct ctacagcaag cttaccgtgt tgggccgcag 1801gtggaccctg gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1861cacgcagaag agcctctccc tgtctccggg taaatctcta gaacaaaaac tcatctcaga 1921agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt gtagccttag 1981acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct 2041aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt 2101ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga 2161gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa 2221aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat 2281taagtgagac cttcgtttgt gcagatccaa catccaaaga cgaaaggttg aatgaaacct 2341ttttgccatc cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg 2401ggatacacta gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac 2461ccacttttgc catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc 2521aattccttct attaggctac taacaccatg actttattag cctgtctatc ctggcccccc 2581tggcgaggtt catgtttgtt tatttccgaa tgcaacaagc tccgcattac acccgaacat 2641cactccagat gagggctttc tgagtgtggg gtcaaatagt ttcatgttcc ccaaatggcc 2701caaaactgac agtttaaacg ctgtcttgga acctaatatg acaaaagcgt gatctcatcc 2761aagatgaact aagtttggtt cgttgaaatg ctaacggcca gttggtcaaa aagaaacttc 2821caaaagtcgg cataccgttt gtcttgtttg gtattgattg acgaatgctc aaaaataatc 2881tcattaatgc ttagcgcagt ctctctatcg cttctgaacc ccggtgcacc tgtgccgaaa 2941cgcaaatggg gaaacacccg ctttttggat gattatgcat tgtctccaca ttgtatgctt 3001ccaagattct ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt 3061ctaaccccta cttgacagca atatataaac agaaggaagc tgccctgtct taaacctttt 3121tttttatcat cattattagc ttactttcat aattgcgact ggttccaatt gacaagcttt 3181tgattttaac gacttttaac gacaacttga gaagatcaaa aaacaactaa ttattcgaaa 3241cgatgagatt tccttcaatt tttactgctg ttttattcgc agcatcctcc gcattagctg 3301ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa gctgtcatcg 3361gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc aacagcacaa 3421ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa gaagaagggg 3481tatctctcga gaaaagagag gctgaagctg aattcgacat tgtgatgacc caggctgcac 3541cctctgtacc tgtcactcct ggagagtcag tatccatctc ctgcaggtct agtaagagtc 3601tcctgcatac taatggcaac acttacttgc attggttcct acagaggcca ggccagtctc 3661ctcagctcct gatatatcgg atgtccgtcc ttgcctcagg agtcccagac aggttcagtg 3721gcagtgggtc aggaactgct ttcacactga gcatcagtag agtggaggct gaggatgtgg 3781gtgtttttta ctgtatgcaa catctagaat atccgctcac gttcggtgct gggaccaagc 3841tggaactgaa acgggctcct cgagaaccac aggtgtacac cctgccccca tcccgggatg 3901agctgggcat cgcgcaagtc agcctgacct gcctggtcaa aggcttctat cccagcgaca 3961tcgccgtgga gtgggagagc aacgggcagc cggagaacaa ctacaagacc acgcctcccg 4021tgctggactc cgacggctct ttcttcctct acagcaagct taccgtgttg ggccgcaggt 4081ggaccctggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac aaccactaca 4141cgcagaagag cctctccctg tctccgggta aagggcgcgc ctccggatgc caaatttacc 4201cgccaaacgc gaacaagatc agagaggctt tgcaatcttg caggaggccc aatgcgcaga 4261gattcggcat atccaactac tgccagatct accccccata cgatgggcgt acaatcatac 4321agcgtgataa cggctatcag cctaactacc acgccgtgaa catcgtcggc tacgagaatg 4381tcgtggttac tgtgaaggta atgggcgatg acggggttct agcttgcgcc atagctacca 4441agtacacttg gaacgtaccc aaaattgcgc cgaaaagtga aaacgtcgta gtgaccataa 4501gggaggcatt ggctcaacct caaagatact gcagacacta ctggacgccc tgcataatcc 4561accgtggtaa accctttcaa cttgaggcag tgttcgaagc taacaggacg gtaacgccaa 4621ttcgtatgca aggtgggtgc gggtcttgtt gggctttttc tggtgtggct gctactgaat 4681aaggcgcgcc tgaggtacct cgagccgcgg cggccgccag ctttctagaa caaaaactca 4741tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta 4801gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct 4861agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt 4921gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct 4981cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt 5041ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac 5101agaagattaa gtgagacctt cgtttgtgcg gatcccccac acaccatagc ttcaaaatgt 5161ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc 5221aaaacaccca agcacagcat actaaatttt ccctctttct tcctctaggg tgtcgttaat 5281tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg 5341aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaattttttt ttttagtttt 5401tttctctttc agtgacctcc attgatattt aagttaataa acggtcttca atttctcaag 5461tttcagtttc atttttcttg ttctattaca acttttttta cttcttgttc attagaaaga 5521aagcatagca atctaatcta aggggcggtg ttgacaatta atcatcggca tagtatatcg 5581gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc 5641cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt 5701tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt 5761tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc 5821gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg 5881cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc 5941gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac 6001ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt cgatatcatg 6061taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag 6121gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 6181ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 6241tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 6301tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 6361gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 6421cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 6481ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 6541tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 6601gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 6661tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 6721ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 6781ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 6841ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 6901accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 6961tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 7021cgttaaggga ttttggtcat gagatc

In the present application, all temperatures are in degrees Celsius. Thefollowing abbreviations are used:

CD32=FcγRII

TLR9=Toll like receptor 9

Der P1=Dermatophagoides pteronissyus major allergen 1

Der P2=Dermatophagoides pteronissyus major allergen 2

Der F1=Dermatophagoides farinae major allergen 1

Example 7: Exemplary Binders 7.1. CD32 Binding Region, Herein AlsoCalled CD32 Binder

The term “CD32 binding region” or “anti-CD32 moiety” as used hereinshall mean a ligand specifically binding to the cellular target CD32,either CD32a, CD32b or both, CD32a and CD32b. The moiety can be anybinding structure, such as derived from proteins, polypeptides orpeptides, including antibodies and antibody fragments or compositemolecules with a binding part. The binding part of the molecules ormolecule complex of the invention can be comprised of proteins such asantibodies or antibody fragments, such as Fab, Fv, VH/VL, scFv, dAb,F(ab)₂, minibody, small mutated immunoglobulin domains, or otherbiological binders, such as soluble T-cell receptor, Darpins, etc.Antibodies and antibody fragments and derivatives may be generated andselected for binding to CD32 according to known methods such ashybridoma technology, B-cell cloning, phage display, ribosome display orcell surface display of antibody libraries, array screening of variantantibodies. Exemplary anti-CD32 moieties are scFv derived from the antiCD32 monoclonal antibody AT-10, IV.3, 2E6 or any other aCD32 monoclonalantibody.

A preferred CD32 binding region is an anti-CD32 antibody or derived froman anti-CD32 antibody, derived from an IgG1 Fc fragment, or a peptidespecifically binding to CD32.

Specifically, the CD32 antibody is selected from the group consisting ofa full-length antibody, an scFv or a VH/VL dimer, specifically bindingthe CD32.

A specific CD32 peptide is a CD32a peptide with the sequence of SEQ ID66.

CD32a Binders:

Antibody specifically binding to CD32a: mAb IV.3 (Stuart et al. (1987)J. Exp. Med. 166: 1668)

ScFV derived from mAb IV.3  (VH-linker-VL): (SEQ ID 67)EVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLE LKGSIUnderlined: VH domain Bold: HL domain Normal type set. Flexible linker (maybe any linker) Anti-CD32a Peptide: Berntzen et al. (J. Biol. Chem. (2009) 284: 1126-1135): (SEQ ID 68): ADGAWAWVWLTETAVGAAK

Group CD32a+b Binders:

Antibody specifically binding to CD32a and CD32b: mAb AT-10 (AbDSerotec)

ScFV derived from mAb AT-10  (VH-linker-VL) (SEQ ID 69):EVKLEESGGGLVQPGGSMKLSCVASGFTFSYYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKNNVYLQMNNLRAEDTGIYYCNRRDEYYAMDYWGQGTSVSVSSGGGGSGGGGSGGGGSDIVLTQSPGSLAVSLGQRATISCRASESVDNFGISFMNWFQQKPGQPPRLLIYGASNQGSGVPARFSGSGSGTDFSLNIHPVEEDDAAMYFCQQSKEVPWTFGGGTKLEI KGSIUnderlined: VH domain Bold: HL domain Normal type set. Flexible linker (maybe any linker) IgG1 Fc fragment (CH2—CH3 domain) (SEQ ID 70):(PKSCDKTHTCPPCP)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKBetween ( ) is hinge region may be omitted Underlined: CH2 domainBold: CH3 domain

7.2 TLR9 Binding Region, Herein Also Called TLR9 Binder or TLR9 Ligand

The term “TLR9 binding region”, “TLR9 binder” or “TLR9 ligand” as usedherein is understood in the following way.

Toll-like receptor 9 (TLR9) recognizes unmethylated bacterial CpG DNAand initiates a signaling cascade leading to the production ofproinflammatory cytokines. There are numerous structures or sequencesthat have been shown to act as a ligand of TLR9, i.e. bind to thisreceptor and thereby either activate (stimulate, upregulate) orde-activate (downregulate) TLR9. For instance, microbial DNA orsynthetic DNA, e.g. synthetic CpG ODN may stimulate TLR9 with variationsin the number and location of CpG dimers, as well as the precise basesequences flanking the CpG dimers. Synthetic CpG ODN differ frommicrobial DNA in that they have a partially or completelyphosphorothioated backbone instead of the typical phosphodiesterbackbone and may or may not have a poly G tail at the 3′ end, 5′ end, orboth.

The TLR9 ligand typically is coupled to the directed adjuvant componentof the present vaccine by chemical coupling e.g. using the commerciallyavailable KIT from Solulink. A peptidic TLR9 ligand may be coupled usingstandard peptide chemistry or may be integrated using recombinant DNAtechnology.

Exemplary TLR9 ligands are ODN 2216 (group 1), ODN 2006/ODN 2007(group2) and CpG-M362 (group 3).

The function of a TLR9 ligand or agonist or antagonist may be determinedin a suitable assay, e.g. in the following way: pDCs are purified fromblood of a healthy donor as described by Tel et al (Immunobiology 2012October; 217(10):1017-24) and subsequently incubated with theappropriate concentration of the TLR9 ligand. After 24 h IFNa ismeasured in the supernatant using standard ELISA protocols. Fordetermination of the maturation state of the cells, pDCs are stained forexpression of CD80 CD83 or CD86 using standard FACS procedures withcommercially available specific antibodies before and after theincubation with the TLR9 ligand.

The number of reactive T cells that are activated upon exposure to thevaccine according to the invention may be determined by a number ofmethods including ELISPOT, FACS analysis, cytokine release, or T cellproliferation assays.

TLR9 ligand is a TLR9 agonist selected from the group consisting of CpGclass A, in particular CpG-A (D)2 oligodeoxynucleotides (ODN), alsoknown as “D”-type ODN. Such TLR9 agonists induce a strong IFNa inductionand minimal maturation of dendritic cells, and are herein called “group1” TLR9 ligand.

According to another aspect of the invention, TLR9 ligand is a TLR9agonist selected from the group consisting of CpG class B, in particularCpG-B (K)2 oligodeoxynucleotides (ODN), also known as “K”-type ODN. SuchTLR9 agonists induce a weak IFNa induction and maturation of dendriticcells, and are herein called “group 2” TLR9 ligand.

According to another aspect of the invention, said TLR9 ligandspecifically is a TLR9 agonist selected from the group consisting of CpGclass C, also known as CpG-C2;3 oligodeoxynucleotides (ODN). Such TLR9agonists induce IFNa and maturation of immature dendritic cells, and areherein called “group 3” TLR9 ligand.

According to another aspect of the invention, TLR9 ligand is a TLR9antagonist selected from the group consisting of inhibitory ODNsoligodeoxynucleotides (sometimes called inhibitory CPGs), e.g. thosewhich contain the inhibitory motif consisting of CCx(not-C)(not-C)xxGGG(x=any base)6. Specific inhibitory ODNs have proven not to induce IFNaand not to induce maturation of dendritic cells, also blockingactivation through an agonist of TLR9.

Such TLR9 agonist or antagonist can be determined in a suitable cellbased assay, which measures stable expression of either of IFNa, or atleast one of the markers CD80, CD83 and CD86, which reflect thematuration of immature dendritic cells (DC). For this purposeplasmacytoid dendritic cells (pDCs) are purified from blood of a healthydonor as described by Tel et al (see above) and subsequently incubatedwith the appropriate concentration of the TLR9 ligand. After 24 h IFNais measured in the supernatant using standard ELISA protocols. Fordetermination of the maturation state of the cells, pDCs are stained forexpression of CD80 CD83 or CD86 using standard FACS procedures withcommercially available specific antibodies before and after theincubation with the TLR9 ligand.

The induction of IFNa may be determined by the level of IFNa expressionand the respective increase with respect to a reference level. Theincrease relative to non-stimulated cells may be compared to theinduction levels induced by established references for each type of CpGas defined by group 1, 2 or 3 TLR9 ligand and is typically between 30%and 300% of the respective reference, preferably at least 100%, morepreferably at least 120%, at least 150%, at least 200% or at least 250%.

The maturation of immature dendritic cells may be determined by thelevel of expression of any of the markers CD80, CD83 and CD86. Therespective increase relative to non-stimulated cells may be compared tothe induction levels induced by established references for each type ofCpG as defined by group 1, 2 or 3 TLR9 ligand and is typically between30% and 300% of the respective reference, preferably at least 100%, morepreferably at least 120%, at least 150%, at least 200% or at least 250%.

Specifically, the TLR9 agonist of group 1 and 3 would result in anincreased IFNa expression and a TRL9 agonist of group 2 and 3 would leadto an increased expression of any of the DC maturation factors CD80,CD83 and CD86. The TLR9 antagonist would result in a reduced IFNaexpression and a reduced expression of any of the DC maturation factorsCD80, CD83 and CD86, even in the presence of a TLR9 agonist of eithergroup 1-3.

CpG Class A

Group CpG-A:

ODN2216: (SEQ ID 71) GGGGGACGATCGTCGGGGGGCpG Class BGroup CpG-B:Natural Ligands:

ODN2006:  (SEQ ID 72) TCGTCGTTTTGTCGTTTTGTCGTTPeptidic Ligands (Peptides):

Name SEQ ID Sequence 12-2 73 ESWDKFLSHYLP  7-6 74 TDWSWFY  7-7 75YPVYWPW   7-12 76 EWWFYWP   7-13 77 WFPIEWW   7-37 78 DQVDIGY   7-38 79THQVYIS 7-12/13 80 WFPIEWWFYWP 12-1 81 DSWQAFLTKFVL 12-3 82 HDIQWFWQHWNS12-4 83 WSWWDHTFNYML 12-6 84 TTQQTWNVRYPY 12-8 85 DHTMPWTRNAKN  12-12 86SWDPYWPFPWFS  12-14 87 AIYYVPSPMFTV  12-16 88 ETTLLKMWLAQM  12-18 89YPWLDVAVVSLY  12-20 90 VPGWHYLATLRA  12-21 91 FDPLGSRDIKGS

As a CpG mimic in the molecule or molecule complex of the invention,such immunostimulatory peptides may be preferably used. Likewisefunctionally active variants thereof may be used, which are fragments,mutants, or hybrids, including combinations thereof.

Functionally active variants are specifically characterized in that theystimulate pDCs, thereby inducing an increased level of IL-6 and/orTNFalpha and/or IFNalpha, as compared to a negative control.

Functionally active variants of the immunostimulatory TLR9 bindingpeptides specifically

a) have at least 60% homology or sequence identity to any of thepeptides of SEQ ID 73-91, preferably at least 70%, at least 80% or atleast 90%;

b) are mutants of any of the peptides of SEQ ID 73-91, obtainable bymodifying the parent amino acid sequence by insertion, deletion orsubstitution of one or more amino acids within the sequence or at eitheror both of the distal ends of the sequence, preferably less than 5, 4,3, 2 or 1 point mutations; or

c) are fragments of any of the peptides of SEQ ID 73-91 comprising atleast 50% of the parent sequence, or at least 60%, at least 70%, atleast 80%, or at least 90%; or at least 5 amino acids, preferably atleast 6, at least 7, at least 8, at least 9, at least 10 or at least 11amino acids.

Specific functional variants comprise a motif selected from the groupconsisting of EWWFYWP (SEQ ID 101), EWW (SEQ ID 102), WFY (SEQ ID 103),YWP (SEQ ID 104), and QVxI, x being any amino acid (SEQ ID 105).

CpG Class C

Group CpG-C

ODNM362: (SEQ ID 92) TCGTCGTCGTTCGAACGACGTTGAT

7.3 Exemplary CD32 Binding Products with Coils

A coiled coil is a structural motif in polypeptides or peptides, inwhich two to seven alpha-helices are coiled together like the strands ofa rope. Such alpha helical regions are likely to form coiled-coilstructures and may be involved in oligomerization of the coil repeats asmeasured in a suitable coiled coil interaction binding assay.

Specifically a dimer of alpha-helices can be formed by contacting thetwo monomers, such that the dimer is formed through an interaction withthe two alpha helix coiled coil domains.

ScFV-coil 1 (IV.3):  (SEQ ID 93)EVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLE LKGSISAWSHPQFEKGPEVSALEKEVSALEKEVSALEKEVSALEKEVSA LEK Underlined: VH domainBold: HL domain Normal type set. Flexible linker (maybe any linker)In italics: pepE coil plus C’ StrepTag II  sequence and “GP”linker may be any flexiblelinker (StrepTag II may be removed or replaced byHIS Tag or any other tag) ScFV-coil 2 (AT10): (SEQ ID 94)EVKLEESGGGLVQPGGSMKLSCVASGFTFSYYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKNNVYLQMNNLRAEDTGIYYCNRRDEYYAMDYWGQGTSVSVSSGGGGSGGGGSGGGGSDIVLTQSPGSLAVSLGQRATISCRASESVDNFGISFMNWFQQKPGQPPRLLIYGASNQGSGVPARFSGSGSGTDFSLNIHPVEEDDAAMYFCQQSKEVPWTFGGGTKLEI KGSISAWSHPQFEKGPEVSALEKEVSALEKEVSALEKEVSALEKEVSAL EK Underlined: VH domainBold: HL domain Normal type set. Flexible linker (maybe any linker)In italics: pepE coil plus at C’ StrepTag II sequence and “GP”linker may be any flexiblelinker (StrepTag II may be removed or replaced byHIS Tag or any other tag) Peptide-coil:  (SEQ ID 95)ADGAWAWVWLTETAVGAAKGPEVSALEKEVSALEKEVSALEKEVSALEK EVSALEKIn italics: pepE coil plus “GP” linker may be any flexible linkerIgG1 Fc fragment-coil:  (SEQ ID 94)(PKSCDKTHTCPPCP)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GPEVSALEKEVSALEKEVSALEKEVSALEKEVSALEK Between ( ) is hinge region may be omittedUnderlined: CH2 domain Bold CH3 domain In italics: pepE coil plus “GP”linker may be any flexible linker

7.4. Exemplary TLR9 Binding Products with SH Group for ChemicalCross-Linking to the CD32 Binder

Group CpG-A:

ODN2216_SH:  (SEQ ID 69) GGGGGACGATCGTCGGGGGG-SH

In bold, flexible linker with SH group for chemical cross-linking toScFV-coil (Maybe any linker and chemically reactive group e.g. NH2suited for chemical crosslinking)

Group CpG-B:

Natural Ligands:

ODN2006_SH:  (SEQ ID 70) TCGTCGTTTTGTCGTTTTGTCGTT-SHPeptidic Ligands_SH:

Name SEQ ID Sequence 12-12_SH 71 SWDPYWPFPWFSGGGS-SH 7-6_SH 72TDWSWFYGGGS-SH 7-7_SH 73 YPVYWPWGGGS-SH 7-12_SH 74 EWWFYWPGGGS-SH7-13_SH 75 WFPIEWWGGGS-SH 7-37_SH 76 DQVDIGYGGGS-SH 7-38_SH 77THQVYISGGGS-SH 7-12/13_SH 78 WFPIEWWFYWPGGGS-SH 12-1_SH 79DSWQAFLTKFVLGGGS-SH 12-2_SH 80 ESWDKFLSHYLPGGGS-SH 12-3_SH 81HDIQWFWQHWNSGGGS-SH 12-4_SH 82 WSWWDHTFNYMLGGGS-SH 12-6_SH 83TTQQTWNVRYPYGGGS-SH 12-8_SH 84 DHTMPWTRNAKNGGGS-SH 12-14_SH 85AIYYVPSPMFTVGGGS-SH 12-16_SH 86 ETTLLKMWLAQMGGGS-SH 12-18_SH 87YPWLDVAVVSLYGGGS-SH 12-20_SH 88 VPGWHYLATLRAGGGS-SH 12-21_SH 89FDPLGSRDIKGSGGGS-SH

In bold, flexible linker with SH group for chemical crosslinking toScFV-coil (Maybe any linker and chemically reactive group e.g. NH2suited for chemical crosslinking)

Group CpG-C

ODNM362_SH:  (SEQ ID 90) TCGTCGTCGTTCGAACGACGTTGAT-SH

In bold flexible linker with SH group for chemical crosslinking toScFV-coil (Maybe any linker and chemically reactive group e.g. NH2suited for chemical crosslinking)

7.5 Exemplary Warhead, i.e. a Structure Comprising a CD32 Binder and aTLR9 Binder

Any representative from the group of CD32 binders chemically linked byany method with any representative of the group of TLR9 binders, wherepreferably the TLR9 binders are coupled to available Lysines (K) in theCD32 binders e.g. Also mixtures of different TLR9 binders may be couplede.g. CpG-B natural or peptidic binders.

ScFV-coil 1 (IV.3) (SEQ ID 91) EVQLQQSGPEL KK PGETV K ISC KASGYTFTNYGMNWV K QAPG K GL K WMG WLNTYTGESIYPDDF K GRFAFSSETSASTAYLQINNLK NEDMATYFCAR GDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSS K SLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGT K LE L K GSISAWSHPQFE KGPEVSALE

EVSALE

EVSALE

EVSAL E

EVSALE

Lysines in coil structure (Italic) are preferred or Peptide-coil:(SEQ ID 93) ADGAWAWVWLTETAVGAA K GPEVSALE

EVSALE

EVSALE

E VSALE

EVSALE

Lysines in coil structure (Italic) are preferred

7.6. Exemplary Immunogen, Herein Also Called Antigen

The term “immunogen” as used herein shall mean one or more antigenstriggering an immune response in a subject. The term “antigen” as usedherein shall in particular refer to any antigenic determinant, which canbe possibly recognized by a binding site of an antibody or is able tobind to the peptide groove of HLA class I or class II molecules and assuch may serve as stimulant for specific T cells. The target antigen iseither recognized as a whole target molecule or as a fragment of suchmolecule, especially substructures, e.g. a polypeptide or carbohydratestructure of targets, generally referred to as “epitopes”, e.g. B-cellepitopes, T-cell epitope), which are immunologically relevant, i.e. arealso recognizable by natural or monoclonal antibodies. Herein the use ofT cell epitopes is preferred.

The term “epitope” as used herein according to the present inventionshall in particular refer to a molecular structure which may completelymake up a specific binding partner or be part of a specific bindingpartner to a binding site of modular antibody of the present invention.The term epitope may also refer to haptens. Chemically, an epitope mayeither be composed of a carbohydrate, a peptide, a fatty acid, anorganic, biochemical or inorganic substance or derivatives thereof andany combinations thereof. If an epitope is a polypeptide, it willusually include at least 3 amino acids, preferably 8 to 50 amino acids,and more preferably between about 10-20 amino acids in the peptide.There is no critical upper limit to the length of the peptide, whichcould comprise nearly the full length of a polypeptide sequence of aprotein. Epitopes can be either linear or conformational epitopes. Alinear epitope is comprised of a single segment of a primary sequence ofa polypeptide or carbohydrate chain. Linear epitopes can be contiguousor overlapping. Conformational epitopes are comprised of amino acids orcarbohydrates brought together by folding of the polypeptide to form atertiary structure and the amino acids are not necessarily adjacent toone another in the linear sequence. Specifically, epitopes are at leastpart of diagnostically relevant molecules, i.e. the absence or presenceof an epitope in a sample is qualitatively or quantitatively correlatedto either a disease or to the health status of a patient or to a processstatus in manufacturing or to environmental and food status. Epitopesmay also be at least part of therapeutically relevant molecules, i.e.molecules which can be targeted by the specific binding domain whichchanges the course of the disease.

One or more epitopes of the same antigen or different antigens may beused according to the present invention, which can include antigens ofall the self-antigens, pathogens, allergens or auto-antigens for whichthe regulation of the immune response is desired, e.g. against whichinduction of a substantial Th1-type response or Treg response (dependingon the type of vaccine) in the host is desired.

In cancer disease an immune response to a self-antigen is desirable. Theterm “self-antigen” as used herein means any antigen, specificallypolypeptide or peptide produced by a normal, healthy subject that doesnot elicit an immune response as such. These self-antigens may beproduced at aberrant or high levels in certain disease states, includingcancer disease, so called tumor associated antigens (TAAs).Self-antigens which are associated with auto-immune disease are hereincalled auto-antigens.

It is understood that the self-antigens can be naturally occurring,recombinantly or synthetically produced. It is also understood that theself-antigens need not be identical to the naturally produced antigen,but rather can include variations thereto having certain homology.

The choice of the self-antigen for use in cancer therapy depends on thetype and stage of the cancer disease, and in particular on theexpression pattern of a cancer cell such as derived from a tumor ormetastases. Specific examples of selected tumor associated antigenspossibly used in a vaccine according to the invention are Epithelialcell adhesion molecule (EpCAM), Lewis Y, alphafetoprotein (AFP) andcarcinoembryonic antigen (CEA), HER2/Neu, VEGF, MUC-1, etc.

The choice of an auto-antigen for use in the therapy of auto-immunediseases depends on the type of the auto-immune disease. Specificexamples of selected auto-immune disease associated antigens possiblyused in a vaccine according to the invention are C1q, ADAMTS13,Desmogelin 3, keratin, gangliosides (e.g. GM1, GD1a, GQ1b), collagentype IV, IgM, cardiolipin, annexin A5, etc.

In some embodiments, the immunogen comprises one or more specificallergens. An “allergen” is an antigen which can initiate a state ofhypersensitivity, or which can provoke an immediate hypersensitivityreaction in a subject already sensitized with the allergen. Allergensare commonly proteins or chemicals bound to proteins which have theproperty of being allergenic. However, allergens can also includeorganic or inorganic materials derived from a variety of synthetic ornatural sources such as plant materials, metals, ingredients incosmetics or detergents, latexes, or the like.

The choice of an allergen for use in the anti-allergy therapy depends onthe type and severity of allergy. Specific examples of selected allergyassociated antigens possibly used in a vaccine according to theinvention are any allergen conventionally used as immunogen,specifically house dust mite allergens (e.g. Der p1, Der p2, Der p3, Derp5, Der p7/ - - - Der p23), cat dander, grass or tree pollen cockroachallergens, etc.

The choice of an antigen specifically inducing immune response against apathogen for use in the prophylaxis or therapy of infectious diseasesdepends on the type of the pathogen, e.g. a microbial or viralinfectious agent. Specific examples of selected pathogen derivedantigens possibly used in a vaccine according to the invention arehepatitis B, hepatitis C, Cholera, HIV, Pertussis, Influenza, Typhoid,etc.

An exemplary antigen is an immunogen comprising one or more T cellepitopes of house dust mite allergens.

Specific antigens are selected from Immunogen 3 comprising the sequenceof position 7-208 of SEQ ID 97, or Immunogen 5-12 comprising thesequence of position 1-364 of SEQ ID 98.

In a different embodiment, an exemplary antigen is a tumor associatedantigen.

Immunogen 3 containing coil (Der P1 and Der P2 Tcell epitopes based on human Class II expression): AA7-208 of SEQ ID 95. (SEQ ID 95)HHHHHHYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGVLACAIATHAKIRDDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWHEIKKVLVPGCHG SEPCIIHRGKPFGGGSGGGSGGKVSALKEKVSALKEKVSALKEKVSALK EKVSALKEUnderlined: HIS tag (may be removed)In bold: a linker (can be any linker)In italics: the pepK coil for interaction with  warheadImmunogen 5-12 containing coil (~29 T cellepitopes of Der pl, Der p2, Der p3, Der p4, Derp7, Der p9, Der p10, Der pll, Der p14, Der p15,based on human Class II expression): AA1-364 of SEQ ID 96. (SEQ ID 96)GVLACAIATHAKIREQERLVKLETVKKSLEQEVRTLHVRIEEVEANALAGGDLRQMRTVTPIRMQGGCGSCWEAHEQQIRIMTTKLKEAEARQQYDIKYTWNVPKIAVNIVGYSNAQGVDYWIVRNSWDTNWYHNPHFIGNRSVITHLMEDLKGELDMRNIQVRGLKQMKRVGDANVKSEDDAFRHYDGRTIIQRDNGYQPNYLDEYWILTAAHCVDGQTVSKLIRSKVLGEKISYYRYVAREQSCRRPNAQRFGISNYCVVVTVKVMGDDELHTYFNVNYTMHYYLNNGATRDILDEYWILTAAHCVAGQTASKLSIRYNSLKHSLFKYRPFKVNELNLEGEFGRELQHKFRLMRNSQMEVEEGGGS HHHHHH GGGSGG KVSALKEKVSALKEKVSALKEKVSALKEKVSALKE Underlined: HIS tag (may be removed)In bold: a linker (can be any linker)In italics: the pepK coil for interaction with warhead

7.7 Exemplary Allergy Vaccine SG100 Against House Dust Mite (HDM)

The exemplary molecule complex is formed by chemical linkage, fusionand/or affinity binding, in particular by a coiled-coil structure.

Warhead (based on ScFV-coil1 IV.3+ODNM362) is mixed with Immunogen 5-12in a ratio which indicates 90% of warhead is complexed with immunogen,no free immunogen (molar ratio of ˜1:1.5) and formulated on Alum.

7.8 Efficacy of SG100 in Rhesus Monkeys

Methods:

5 healthy house dust mite (HDM) naïve rhesus monkeys were immunized 3×with SG100 (100 μg/shot) absorbed on Alum) on d0, d14 and d28. Bloodsamples were taken on d0 and d49 for T cell activation and antibodyproduction.

Antibody Immune Response:

Serum samples were tested in standard ELISA for IgG antibodies againstwarhead, immo 5-12 (Immo5), Der p1, Der p2,Der p5 and Der p7. Theantigens were coated to maxisorb plates (1 μg/ml I PBS) overnight at 4°C., washed twice, blocked with PBS 1% BSA, washed twice incubated withthe sera in a 1:1000 dilution for 1 h at 4° C., washed twice andsubsequently detected with anti-human-IgG-PO (cross reactive with rhesusmonkey IgG).

Cellular Immune Response:

Proliferation: PBMC (105/well) were cultured for 4 days (37° C./5%CO2/99% humidity) in 8 plex with medium, warhead (2 μg/ml), Immunogen (2μg/ml), Der p1 (2 μg/ml), Der p2 (2 μg/ml), Der p5 (2 μg/ml) and Der p7(2 μg/ml). As positive control Con A (Concanavaline A, Sigma) was used.Proliferation was measured by [3H]-thymidine (0.5 μCi/well) during thefinal 18 hrs of a 4 day culture. Cells were harvested forβ-scintillation counting (Topcount NXT, Packerd, Ramsey, Minn., USA).Net counts per minutes were calculated by subtracting the counts of themedium control from the counts induced by the different antigens.

Cytokine Production:

From each well of the 8plex stimulations of the proliferationexperiment, 50 μl supernatant was taken after 24 h and pooled. Thepooled supernatants were tested for the presence of IFN and IL-4 usingcommercially available ELISA kits from U-Cytech, Utrecht TheNetherlands).

Results:

Antibody Responses:

Strong IgG responses were measured against the warhead and the immunogenof SG100, but no antibodies were detected against Der p1, Der p2, Der p5or Der p7 (FIG. 5), indicating that the animals were naive for thetested HDM allergens and that SG100 does not contain B cell epitopes,which cross-react with the tested HDM allergens.

T Cell Response:

In FIG. 6 it can be seen that PBMCs of all treated animals showed strongproliferation when stimulated in vitro with warhead, immo5, Der p1, Derp2, Der p′7, but not against Der p5. Also IFN but no IL-4 was measuredin supernatants from in vitro cultures with warhead, immo5, Der p1, Derp2, Der p7 but not against Der p5. (FIG. 7). IL-4 was seen afterstimulation with Con A (data not shown).

Conclusion:

Immunization with SG100 induces a Th1 type memory response against thevaccine as indicated by the presence of IgG antibodies as well theinduction of T cells which produce IFN but not IL-4 when stimulated bywarhead or Immo5. As expected, no IgG (=B cell memory) against Der p1,Der p2, Der p5 or Der p7 was induced because the vaccine does notcontain B ell epitopes from these allergens. However, Th1 type memory,was induced against the T cell epitopes of the house dust mite allergenswhich are present in the vaccine Der p1, Der p2, Der p7. No Th1 typememory is induced against Der p5, which is not included in the vaccine.This confirms the concept of SG100.

7.7 Exemplary Vaccine, Warhead for Use in Oncology

ScFV-coil 1 (IV.3): (SEQ ID 93)EVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLE LKGSISAWSHPQFEKGPEVSALEKEVSALEKEVSALEKEVSALEKEVSA LEK Underlined: VH domainBold: HL domain Normal type set. Flexible linker (maybe any linker)In italics: pepE coil plus C’ StrepTag II sequence and “GP”linker may be any flexiblelinker (StrepTag II may be removed or replaced byHIS Tag or any other tag)

Warhead with ODNM362 (SEQ ID NO:93):

EVQLQQSGPEL KK PGETV K ISC K ASGYTFTNYGMNWV K QAPG K GL K WMGWLNTYTGESIYPDDF K GRFAFSSETSASTAYLQINNL K NEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVP VTPGESVSISCRSS KSLLHTNGNTYLHWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTAFTLSISRVEAEDVGVFYCMQHLEYPLTFGAGT K LE L K GSISAWSHPQFE KGPEVSALE

EVSALE

EVSALE

EVSALE

EVSALE

ODNM362_SH (SEQ ID 92) TCGTCGTCGTTCGAACGACGTTGAT-SHODN-M362 may be coupled to any of the available lysines in ScFV-1-coil

Warhead (SG100):

ScFV-1-coil chemically linked with ODN-M362-SH. The preparation is a mixof ScFV-1-coil linked with 1 to 18 molecules ODN-M362 preferred is a mixwith 1-6 molecules ODN-M362 coupled to ScFV-1-coil. All these mixes maybe named warhead or ScFv-1-coil-M362.

Background:

Oncological targets for active immunotherapy are almost per definitionautoantigens which are over expressed on tumor cells. These antigens arecalled tumor associated antigens (TAA) and the immune system is not ableto respond against these antigens because they are recognized as self. Avaccine formulation that enables the immune system to generate aspecific antibody and/or cellular immune response against an autoantigenis potentially suited for use as anti-tumor vaccination.

The Warhead of SG100 Enables an Autoimmunresponse:

24 mice (6/group) were immunized s.c. 2× with 35 μg in 150 μlScFV-1-coil or with warhead (ScFV-1-coil-M362) either formulated on Alumor diluted in PBS. Immunizations were done on d0, and d14, sera weretaken on d0 (before immunization) and d28 and analyzed for IgG1 andIgG2a against ScFV-1-coil (indicated as ScFV) and mAb IV.3 by standardELISA. See FIG. 1.

As can be seen in FIG. 1, immunization with warhead induced a strongIgG1 and IgG2 response to ScFV-1-coil as well as to mAb IV.3 on day 28.A positive response was seen independent of the presence of Alum.Immunization with ScFV-1-coil only induced an IgG1 response againstScFV-1-coil and only in the presence of Alum, no IgG2a response wasinduced. These data fit with the concept that CpG (M362) induces a Th1type response (IgG2a) and Alum induces a TH2 type response (IgG1). Theresponse against ScFV-1-coil indicates that this protein is immunogenicin the mouse, indeed both the StrepTagII (amino acid sequence“SAWSHPQFEK” (SEQ ID 97)) and the pepE (amino acid sequence“EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK” SEQ ID 100) were target of the IgGresponses (data not shown). However ScFV-1-coil also contains“mouse-self-sequences” because the ScFV contains the VH and VL domainsof the mouse mAb IV.3. Therefore, an immune response against IV.3indicates the presence of autoimmune antibodies. Indeed, only thewarhead with or without Alum was able to induce this type of immuneresponse. Hence, the presence of M362 on the ScFV-1-coil is able tobreak the tolerance against the autoantigens VH and VL domain of theparent antibody. By combining an autoantigen e.g. a TAA, through highaffinity interaction with pepE of the warhead, the warhead will be ableto induce the necessary autoimmune response against the TAA. The complexof the warhead (ScFV-1-coil-M362) with the TAA forms a potent vaccinefor the treatment of cancer with over expression of the TAA in thevaccine. Such a vaccine may be formulated with any adjuvants, e.g. onAlum.

Example 8: Comparison of the Stimulatory Capacity of the TLR9 Binder CPGon Human Plasmocytoid Dendritic Cells Administered in a Complex with anAnti-CD32 Antibody and in a Mixture 8.1 Material and Methods

a) Plasmacytoid Dendritic (pDCs) cells:

Buffy coats were obtained from healthy volunteers according toinstitutional guidelines and pDCs were purified by positive isolationusing anti-BDCA-4-conjugated magnetic microbeads (Miltenyi Biotec) andcultured in X-VIVO-15 medium (Cambrex) supplemented with 2% ofdecomplemented human AB+ serum. pDCs purity was routinely up to 95%, asassessed by double staining BDCA-2/CD123 (Miltenyi Biotec) in a FACS.

b) Stimulation of pDCs:

Freshly isolated pDCs were incubated with biotinylated anti-CD32 (10μg/ml, clone AT10, AbD Serotec) in PBA (PBS containing 5% BSA) on icefor 30 minutes and washed twice with PBA, followed by an incubation with10 μg/ml streptavidin-Alexa647 in PBA on ice for 30 minutes and twotimes washing with PBA. Subsequently, pDCs were incubated on ice for 15minutes with either PBA, 5 μg/ml ODN-CpG C (M362, Axorra) in PBA or 5μg/ml ODN-CpG C-3′-biotin in PBA (M362, Biosearch Technologies). UnboundODN-CpG C was washed away three time with PBA and pDCs were culturedovernight (37° C., 5% CO2) in X-VIVO-15 medium (Cambrex), supplementedwith 2% of decomplemented human AB+ serum. Supernatants were collectedfrom pDC cultures after overnight stimulation, and IFNα production wasanalyzed with murine monoclonal capture and HRP-conjugated anti-IFNaantibodies (BenderMed systems) using standard ELISA procedures.

8.2. Results

pDCs stimulated with 5 μg/ml CPG-C+aCD32-biotin/streptavidin-Alexa647produced significantly more IFN-α, than pDCs stimulated withaCD32-biotin/streptavidin-Alexa647 alone. When aCD32 and CPG-C-biotinwere complexed into one molecule, there was a statistically significant(p<0.001) positive synergistic effect on IFNα production compared toeither aCD32-biotin/streptavidin-Alexa647 alone oraCD32-biotin/streptavidin-Alexa647+5 μg/ml CPG-C non-complexed. (FIG.2). In a control experiment, there was no significant difference in IFNαproduction, when pDCs were stimulated with CPG-C versus CPG-biotin orCPG-C versus CPG-C+aCD32-biotin/streptavidin-Alexa647 (data not shown).

FIG. 2: IFNα Production of pDCS after Stimulation with CPG-C.

Maximum IFNα production in each experiment was seen when CPG-C-biotinwas complexed with aCD32biotin through streptavidin. This was set at100% and the ratio was calculated for aCD32-biotin with or without CPG-C(non complexed). Statistical analyses was done with the paired studentt-test; p<0.05 were considered significant.

Example 9: Effect of a Molecule Complex Comprising Different Formats ofAnti-CD32 And TLR9 Binding Moieties on the Immune Response as Determinedon Plasmacytoid Dendritic (pDCs) Cells Stimulation of pDCs with CpGTargeted to CD32, Employing an Anti-CD32 Single Chain Antibody 9.1Material and Methods

a) Plasmacytoid Dendritic (pDCs) cells:

Buffy coats were obtained from healthy volunteers according toinstitutional guidelines and pDCs were purified by positive isolationusing anti-BDCA-4-conjugated magnetic microbeads (Miltenyi Biotec) andcultured in X-VIVO-15 medium (Cambrex) supplemented with 2% ofdecomplemented human AB+ serum. pDCs purity was routinely up to 95%, asassessed by double staining BDCA-2/CD123 (Miltenyi Biotec) in a FACS.

b) Stimulation of pDCs:

Freshly isolated pDCs were incubated with biotinylated anti-CD32 (10μg/ml, clone AT10, AbD Serotec; ScFV from IV.3) in PBA (PBS containing5% BSA) on ice for 30 minutes and washed twice with PBA, followed by anincubation with 10 μg/ml streptavidin-Alexa647 in PBA on ice for 30minutes and two times washing with PBA. Subsequently, pDCs wereincubated on ice for 15 minutes with either PBA, 5 μg/ml ODN-CpG C(M362, Axorra) in PBA or 5 μg/ml ODN-CpG C-3′-biotin in PBA (M362,Biosearch Technologies). Unbound ODN-CpG C was washed away three timewith PBA and pDCs were cultured overnight (37° C., 5% CO2) in X-VIVO-15medium (Cambrex), supplemented with 2% of decomplemented human AB+serum. Supernatants were collected from pDC cultures after overnightstimulation, and IFNα production was analyzed with murine monoclonalcapture and HRP-conjugated anti-IFNα antibodies (BenderMed systems)using standard ELISA procedures.

9.2. Results

IFNα production of pDCS after stimulation with CPG-C: Maximum IFNαproduction in each experiment was seen when CPG-C-biot was complexedwith aCD32biot through streptavidin. Stimulation with CpG withouttargeting (CpG standard) was significantly less potent than CpG targetedto CD32 (p<0.04 resp. p<0.02). There is no difference when the aCD32antibody AT10 (specific for CD32a and CD32b) was used or whether anunrelated single chain antibody (specific for CD32a only) was used. Thisconfirms that the enhancement is independent of the epitope that isrecognized on CD32 and independent of the type of binder that is used.Statistical analyses was done with the paired student t-test; p<0.05were considered significant (see FIG. 3).

Example 10: Effect of a Molecule Complex Comprising Different Formats ofAnti-CD32 And TLR9 Binding Moieties on the Immune Response as Determinedon Plasmacytoid Dendritic (pDCs) Cells Stimulation of pDCs with CpGTargeted to CD32, Employing an aCD32 Peptide, Read Out IL-6 and TNFαProduction 10.1 Material and Methods

a) PBMC Cells:

Buffy coats were obtained from the Red Cross Austria. Cells wereseparated on ficoll hypack.

b) Stimulation of pDCs:

Freshly isolated PBMC's were incubated with 200 μg/ml aCD32a peptidepublished by Berntzen et al (J. Biol. Chem. (2009) 284:1126-1135;sequence ADGAWAWVWLTETAVGAAK-biotin, SEQ ID NO:68) in PBA (PBScontaining 5% BSA) on ice for 30 minutes and washed twice with PBA,followed by an incubation with 10 μg/ml streptavidin in PBA on ice for30 minutes and two times washing with PBA. Subsequently, PBMC's wereincubated on ice for 15 minutes with either PBA, 5 μg/ml ODN-CpG C(M362, Axorra) in PBA or 5 μg/ml ODN-CpG C-3′-biotin in PBA (M362,Girindus). Unbound ODN-CpG C was washed away three time with PBA andPBMCs were cultured overnight (37° C., 5% CO2) in X-VIVO-15 medium(Cambrex), supplemented with 2% of decomplemented human AB+ serum.Supernatants were collected from PBMC cultures after overnightstimulation, and IFN^(L) production was analyzed with murine monoclonalcapture and HRP-conjugated anti-IL-6 or TNF^(L) antibodies (BenderMedsystems) using standard ELISA procedures.

10.2. Results

IL-6 production of pDCS in PBMC after stimulation with CPG-C: MaximumIL-6 production was seen when CPG-C-biot was complexed with aCD32biotthrough streptavidin. Stimulation with CpG without targeting (CpGstandard) was ˜75 times less potent than CpG targeted to CD32. Whereasfor TNFα the induction was ˜3 times stronger when CpG was targeted toCD32. This confirms that the enhancement is independent of the epitopethat is recognized on CD32 and independent of the type of binder that isused. Statistical analyses was done with the paired student t-test;p<0.05 were considered significant (see FIG. 4).

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. The steps disclosed for the present methods, for example, arenot intended to be limiting nor are they intended to indicate that eachstep is necessarily essential to the method, but instead are exemplarysteps only. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure.

Recitation of value ranges herein is merely intended to serve as ashorthand method for referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All references cited herein areincorporated by reference in their entirety.

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The invention claimed is:
 1. A composition which comprises a molecule ormolecule complex comprising a TLR9 binding part capable of binding toTLR9, a CD32 binding part capable of binding to CD32, and at least oneantigen, wherein the CD32 binding part is selected from the groupconsisting of antibodies and CD32-binding antibody fragments, andwherein the TLR9 binding part is a CpG oligodeoxynucleotide.
 2. Thecomposition of claim 1, characterized in that the antigen is an allergenor part of an allergen.
 3. The composition of claim 1, characterized inthat the antigen is non-covalently linked to the molecule or moleculecomplex.
 4. The composition of claim 1, characterized in that theantigen is non-covalently linked to the TLR9 binding part or the CD32binding part.
 5. The composition of claim 2, characterized in that theallergen is selected from the group consisting of an allergen associatedwith atopic dermatitis, an allergen associated with allergic asthma, anallergen associated with allergic rhinitis or an allergen associatedwith allergic conjunctivitis.
 6. The composition of claim 1,characterized in that the antigen is selected from the group consistingof a denatured antigen and an antigen modified to prevent binding toIgE.
 7. The composition of claim 1, wherein the CD32-binding antibodyfragment is an scFv of an antibody.
 8. The composition of claim 1,characterized in that the antibody is selected from the group consistingof IgG, IgM, IgE, IgA and IgD.
 9. The composition of claim 1, whereinthe antibody is selected from the group consisting of a human antibodyand a humanized antibody.
 10. The composition of claim 1, characterizedin that at least part of the antibody is derived from a mammal selectedfrom the group consisting of a human, a mouse, and a camel.
 11. Thecomposition of claim 1, further comprising at least one pharmaceuticallyacceptable carrier or diluent.
 12. The composition of claim 1, whereinthe TLR9 binding part is any of CpG-A, CpG-B or CpG-C ligands.
 13. Thecomposition of claim 1, wherein the antigen comprises one or more T cellepitopes of house dust mite allergens.
 14. The composition of claim 13,wherein the antigen is selected from the group consisting of Immunogen 3comprising the sequence of positions 7-208 of SEQ ID NO:97 and Immunogen5-12 comprising the sequence of positions 1-364 of SEQ ID NO:98.
 15. Thecomposition of claim 1, wherein the antigen is a tumor associatedantigen.